Compositions and methods for producing isoprene

ABSTRACT

The invention features methods for producing isoprene from cultured cells. The invention also provides compositions that include these cultured cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 12/335,071, filed Dec. 15, 2008, which is now U.S. Pat. No.8,288,148, which claims priority benefit of U.S. Provisional ApplicationNo. 61/013,574, filed Dec. 13, 2007. The disclosures of which are hereinincorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to methods for producingisoprene from cultured cells and compositions that include thesecultured cells.

BACKGROUND OF THE INVENTION

Isoprene (2-methyl-1,3-butadiene) is the critical starting material fora variety of synthetic polymers, most notably synthetic rubbers.Isoprene is naturally produced by a variety of microbial, plant, andanimal species. In particular, two pathways have been identified for thebiosynthesis of isoprene: the mevalonate (MVA) pathway and thenon-mevalonate (DXP) pathway (FIGS. 19A and 19B). However, the yield ofisoprene from naturally-occurring organisms is commerciallyunattractive. About 800,000 tons per year of cis-polyisoprene areproduced from the polymerization of isoprene; most of this polyisopreneis used in the tire and rubber industry. Isoprene is also copolymerizedfor use as a synthetic elastomer in other products such as footwear,mechanical products, medical products, sporting goods, and latex.

Currently, the tire and rubber industry is based on the use of naturaland synthetic rubber. Natural rubber is obtained from the milky juice ofrubber trees or plants found in the rainforests of Africa. Syntheticrubber is based primarily on butadiene polymers. For these polymers,butadiene is obtained as a co-product from ethylene and propylenemanufacture.

While isoprene can be obtained by fractionating petroleum, thepurification of this material is expensive and time-consuming. Petroleumcracking of the C5 stream of hydrocarbons produces only about 15%isoprene. Thus, more economical methods for producing isoprene areneeded. In particular, methods that produce isoprene at rates, titers,and purity that are sufficient to meet the demands of a robustcommercial process are desirable. Also desired are systems for producingisoprene from inexpensive starting materials.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the invention features cells in culture that produceisoprene. In some embodiments, the invention provides cells in culturethat produce greater than about 400 nmole of isoprene/gram of cells forthe wet weight of the cells/hour (nmole/g_(wcm)/hr) of isoprene. In someembodiments, the cells have a heterologous nucleic acid that (i) encodesan isoprene synthase polypeptide and (ii) is operably linked to apromoter. In some embodiments, the cells are cultured in a culturemedium that includes a carbon source, such as, but not limited to, acarbohydrate (e.g., xylose or glucose), acetate, glycerol, glycerine,dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fattyacid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride,triglyceride, renewable carbon source (e.g., a hydrolyzed biomass carbonsource), polypeptide (e.g., a microbial or plant protein or peptide),yeast extract, component from a yeast extract, or any combination of twoor more of the foregoing.

In some embodiments, the invention provides cells in culture that havean average volumetric productivity of isoprene greater than about 0.1mg/L_(broth)/hr. In some embodiments, the invention provides cells inculture that have a peak volumetric productivity of isoprene greaterthan about 0.5 mg/L_(broth)/hr. In some embodiments, the cells have aheterologous nucleic acid that (i) encodes an isoprene synthasepolypeptide and (ii) is operably linked to a promoter. In someembodiments, the cells are cultured in a culture medium that includes acarbon source, such as, but not limited to, a carbohydrate (e.g., xyloseor glucose), acetate, glycerol, glycerine, dihydroxyacetone, one-carbonsource, oil, animal fat, animal oil, fatty acid, lipid, phospholipid,glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbonsource (e.g., a hydrolyzed biomass carbon source), polypeptide (e.g., amicrobial or plant protein or peptide), yeast extract, component from ayeast extract, or any combination of two or more of the foregoing.

In some embodiments, the invention provides cells in culture thatconvert more than about 0.002% of the carbon in a cell culture mediuminto isoprene. In some embodiments, the cells have a heterologousnucleic acid that (i) encodes an isoprene synthase polypeptide and (ii)is operably linked to a promoter. In some embodiments, the cells arecultured in a culture medium that includes a carbon source, such as, butnot limited to, a carbohydrate (e.g., xylose or glucose), acetate,glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animalfat, animal oil, fatty acid, lipid, phospholipid, glycerolipid,monoglyceride, diglyceride, triglyceride, renewable carbon source (e.g.,a hydrolyzed biomass carbon source), polypeptide (e.g., a microbial orplant protein or peptide), yeast extract, component from a yeastextract, or any combination of two or more of the foregoing.

In some embodiments, the invention provides cells in culture thatcomprise a heterologous nucleic acid encoding an isoprene synthasepolypeptide. In some embodiments, the cells have a heterologous nucleicacid that (i) encodes an isoprene synthase polypeptide and (ii) isoperably linked to a promoter. In some embodiments, the cells arecultured in a culture medium that includes a carbon source, such as, butnot limited to, a carbohydrate (e.g., xylose or glucose), acetate,glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animalfat, animal oil, fatty acid, lipid, phospholipid, glycerolipid,monoglyceride, diglyceride, triglyceride, renewable carbon source (e.g.,a hydrolyzed biomass carbon source), polypeptide (e.g., a microbial orplant protein or peptide), yeast extract, component from a yeastextract, or any combination of two or more of the foregoing.

In one aspect, the invention features methods of producing isoprene,such as methods of using any of the cells described herein to produceisoprene. In some embodiments, the method involves culturing cells underconditions sufficient to produce greater than about 400 nmole/g_(wcm)/hrof isoprene. In some embodiments, the method also includes recoveringisoprene produced by the cells. In some embodiments, the method includespurifying isoprene produced by the cells. In some embodiments, themethod includes polymerizing the isoprene. In some embodiments, thecells have a heterologous nucleic acid that (i) encodes an isoprenesynthase polypeptide and (ii) is operably linked to a promoter. In someembodiments, the cells are cultured in a culture medium that includes acarbon source, such as, but not limited to, a carbohydrate (e.g., xyloseor glucose), acetate, glycerol, glycerine, dihydroxyacetone, one-carbonsource, oil, animal fat, animal oil, fatty acid, lipid, phospholipid,glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbonsource (e.g., a hydrolyzed biomass carbon source), polypeptide (e.g., amicrobial or plant protein or peptide), yeast extract, component from ayeast extract, or any combination of two or more of the foregoing.

In one aspect, the invention features methods of producing isoprene,such as methods of using any of the cells described herein to produceisoprene. In some embodiments, the method involves culturing cells underconditions resulting in an average volumetric productivity of isoprenegreater than about 0.1 mg/L_(broth)/hr. In some embodiments, the methodinvolves culturing cells under conditions resulting in a peak volumetricproductivity of isoprene greater than about 0.5 mg/L_(broth)/hr. In someembodiments, the method also includes recovering isoprene produced bythe cells. In some embodiments, the method includes purifying isopreneproduced by the cells. In some embodiments, the method includespolymerizing the isoprene. In some embodiments, the cells have aheterologous nucleic acid that (i) encodes an isoprene synthasepolypeptide and (ii) is operably linked to a promoter. In someembodiments, the cells are cultured in a culture medium that includes acarbon source, such as, but not limited to, a carbohydrate (e.g., xyloseor glucose), acetate, glycerol, glycerine, dihydroxyacetone, one-carbonsource, oil, animal fat, animal oil, fatty acid, lipid, phospholipid,glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbonsource (e.g., a hydrolyzed biomass carbon source), polypeptide (e.g., amicrobial or plant protein or peptide), yeast extract, component from ayeast extract, or any combination of two or more of the foregoing.

In some embodiments, the method includes culturing cells underconditions sufficient to convert more than about 0.002% of the carbon ina cell culture medium into isoprene. In some embodiments, the methodalso includes recovering isoprene produced by the cells. In someembodiments, the method includes purifying isoprene produced by thecells. In some embodiments, the method includes polymerizing theisoprene. In some embodiments, the cells have a heterologous nucleicacid that (i) encodes an isoprene synthase polypeptide and (ii) isoperably linked to a promoter. In some embodiments, the cells arecultured in a culture medium that includes a carbon source, such as, butnot limited to, a carbohydrate (e.g., xylose or glucose), acetate,glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animalfat, animal oil, fatty acid, lipid, phospholipid, glycerolipid,monoglyceride, diglyceride, triglyceride, renewable carbon source (e.g.,a hydrolyzed biomass carbon source), polypeptide (e.g., a microbial orplant protein or peptide), yeast extract, component from a yeastextract, or any combination of two or more of the foregoing.

In some embodiments of any of the aspects of the invention, the cells inculture produce isoprene at greater than or about 400, 500, 600, 700,800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000,10,000, 12,500, 20,000, 30,000, 40,000, 50,000, 75,000, 100,000,125,000, 150,000, 188,000, or more nmole/g_(wcm)/hr isoprene. In someembodiments, the cells in culture have an average volumetricproductivity of isoprene at greater than or about 0.1, 1.0, 10, 25, 50,100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1100,1200, 1300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100,2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100,3,200, 3,300, 3,400, 3,500, or more mg of isoprene/L of broth/hr(mg/L_(broth)/hr, wherein the volume of broth includes the volume of thecells and the cell medium). In some embodiments, the cells in culturehave a peak volumetric productivity of isoprene at greater than or about0.5, 1.0, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800,900, 1,000, 1100, 1200, 1300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900,2,000, 2,100, 2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900,3,000, 3,100, 3,200, 3,300, 3,400, 3,500, 3,750, 4,000, 4,250, 4,500,4,750, 5,000, 5,250, 5,500, 5,750, 6,000, 6,250, 6,500, 6,750, 7,000,7,250, 7,500, 7,750, 8,000, 8,250, 8,500, 8,750, 9,000, 9,250, 9,500,9,750, 10,000, 12,500, 15,000, or more mg of isoprene/L of broth/hr(mg/L_(broth)/hr, wherein the volume of broth includes the volume of thecells and the cell medium). In some embodiments of any of the aspects ofthe invention, the cells in culture convert greater than or about 0.002,0.005, 0.01, 0.02, 0.05, 0.1, 0.12, 0.14, 0.16, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 2.0, 2.2, 2.4, 2.6, 3.0, 4.0, 5.0,6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0,18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 23.2, 23.4, 23.6, 23.8, 24.0, 25.0,30.0, 31.0, 32.0, 33.0, 35.0, 37.5, 40.0, 45.0, 47.5, 50.0, 55.0, 60.0,65.0, 70.0, 75.0, 80.0, 85.0, 90.0 molar %, or more of the carbon in thecell culture medium into isoprene. In some embodiments of any of theaspects of the invention, the cells in culture produce isoprene atgreater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500,600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000,4,000, 5,000, 10,000, 100,000, or more ng of isoprene/gram of cells forthe wet weight of the cells/hr (ng/g_(wcm)/h). In some embodiments ofany of the aspects of the invention, the cells in culture produce acumulative titer (total amount) of isoprene at greater than or about 1,10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900,1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000,50,000, 100,000, or more mg of isoprene/L of broth (mg/L_(broth),wherein the volume of broth includes the volume of the cells and thecell medium). Other exemplary rates of isoprene production and totalamounts of isoprene production are disclosed herein.

In some embodiments of any of the aspects of the invention, the cellsfurther comprise a heterologous nucleic acid encoding an IDIpolypeptide. In some embodiments of any of the aspects of the invention,the cells further comprise an insertion of a copy of an endogenousnucleic acid encoding an IDI polypeptide. In some embodiments of any ofthe aspects of the invention, the cells further comprise a heterologousnucleic acid encoding a DXS polypeptide. In some embodiments of any ofthe aspects of the invention, the cells further comprise an insertion ofa copy of an endogenous nucleic acid encoding a DXS polypeptide. In someembodiments of any of the aspects of the invention, the cells furthercomprise one or more nucleic acids encoding an IDI polypeptide and a DXSpolypeptide. In some embodiments of any of the aspects of the invention,one nucleic acid encodes the isoprene synthase polypeptide, IDIpolypeptide, and DXS polypeptide. In some embodiments of any of theaspects of the invention, one vector encodes the isoprene synthasepolypeptide, IDI polypeptide, and DXS polypeptide. In some embodiments,the vector comprises a selective marker, such as an antibioticresistance nucleic acid.

In some embodiments of any of the aspects of the invention, theheterologous isoprene synthase nucleic acid is operably linked to a T7promoter, such as a T7 promoter contained in a medium or high copyplasmid. In some embodiments of any of the aspects of the invention, theheterologous isoprene synthase nucleic acid is operably linked to a Trcpromoter, such as a Trc promoter contained in a medium or high copyplasmid. In some embodiments of any of the aspects of the invention, theheterologous isoprene synthase nucleic acid is operably linked to a Lacpromoter, such as a Lac promoter contained in a low copy plasmid. Insome embodiments of any of the aspects of the invention, theheterologous isoprene synthase nucleic acid is operably linked to anendogenous promoter, such as an endogenous alkaline serine proteasepromoter. In some embodiments, the heterologous isoprene synthasenucleic acid integrates into a chromosome of the cells without aselective marker.

In some embodiments of any of the aspects of the invention, at least aportion of the cells maintain the heterologous isoprene synthase nucleicacid for at least or about 5, 10, 20, 40, 50, 60, 65, or more celldivisions in a continuous culture (such as a continuous culture withoutdilution). In some embodiments of any of the aspects of the invention,the nucleic acid comprising the isoprene synthase, IDI, or DXS nucleicacid also comprises a selective marker, such as an antibiotic resistancenucleic acid.

In some embodiments of any of the aspects of the invention, the cellsfurther comprise a heterologous nucleic acid encoding an MVA pathwaypolypeptide (such as an MVA pathway polypeptide from Saccharomycescerevisi, Methanosarcina mazei, or Enterococcus faecalis). In someembodiments of any of the aspects of the invention, the cells furthercomprise an insertion of a copy of an endogenous nucleic acid encodingan MVA pathway polypeptide (such as an MVA pathway polypeptide fromSaccharomyces cerevisia, Methanosarcina mazei, or Enterococcusfaecalis). In some embodiments of any of the aspects of the invention,the cells comprise an isoprene synthase, DXS, and MVA pathway nucleicacid. In some embodiments of any of the aspects of the invention, thecells comprise an isoprene synthase nucleic acid, a DXS nucleic acid, anIDI nucleic acid, and a MVA pathway nucleic (in addition to the IDInucleic acid).

In some embodiments of any of the aspects of the invention, the isoprenesynthase polypeptide is a naturally-occurring polypeptide from a plantsuch as Pueraria (e.g., Pueraria montana) or Populus (e.g., Populustremuloides, Populus alba (P. alba), Populus nigra, Populus trichocarpa,or the hybrid, Populus alba×Populus tremula).

In some embodiments of any of the aspects of the invention, the cellsare bacterial cells, such as gram-positive bacterial cells (e.g.,Bacillus cells such as Bacillus subtilis cells or Streptomyces cellssuch as Streptomyces lividans, Streptomyces coelicolor, Streptomycescoelicolor, Streptomyces albus, or Streptomyces griseus cells). In someembodiments of any of the aspects of the invention, the cells aregram-negative bacterial cells (e.g., Escherichia cells such asEscherichia coli cells or Pantoea cells such as Pantoea citrea cells).In some embodiments, the E. coli cells are E. coli FadR atoC mutantcells. In some embodiments, the E. coli cells express (such asconstitutively express) ybhE (also known as pgl). In some embodiments ofany of the aspects of the invention, the cells are fungal, cells such asfilamentous funal cells (e.g., Trichoderma cells such as Trichodermareesei cells or Aspergillus cells such as Aspergillus oryzae andAspergillus niger) or yeast cells (e.g., Yarrowia cells such as Yarrowialipolytica cells).

In some embodiments of any of the aspects of the invention, themicrobial polypeptide carbon source includes one or more polypeptidesfrom yeast or bacteria. In some embodiments of any of the aspects of theinvention, the plant polypeptide carbon source includes one or morepolypeptides from soy, corn, canola, jatropha, palm, peanut, sunflower,coconut, mustard, rapeseed, cottonseed, palm kernel, olive, safflower,sesame, or linseed.

In one aspect, the invention provides a tire comprising polyisoprene. Insome embodiments, the polyisoprene is produced by (i) polymerizingisoprene from any of the compositions or methods described herein or(ii) polymerizing isoprene recovered from any of the compositions ormethods described herein. In some embodiments, the polyisoprenecomprises cis-1,4-polyisoprene.

In one aspect, the invention features a product (such as a tire)produced by any of the compositions or methods of the invention.

It is to be understood that one, some, or all of the properties of thevarious embodiments described herein may be combined to form otherembodiments of the present invention. These and other aspects of theinvention will become apparent to one of skill in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the nucleotide sequence of a kudzu isoprene synthase genecodon-optimized for expression in E. coli (SEQ ID NO:1). The atg startcodon is in italics, the stop codon is in bold and the added PstI siteis underlined.

FIG. 2 is a map of pTrcKudzu.

FIG. 3 is the nucleotide sequence of pTrcKudzu (SEQ ID NO:2). The RBS isunderlined, the kudzu isoprene synthase start codon is in bold capitolletters and the stop codon is in bold, capitol, italics letters. Thevector backbone is pTrcHis2B.

FIG. 4 is a map of pETNHisKudzu.

FIG. 5 is the nucleotide sequence of pETNHisKudzu (SEQ ID NO:5).

FIG. 6 is a map of pCL-lac-Kudzu.

FIG. 7 is the nucleotide sequence of pCL-lac-Kudzu (SEQ ID NO:7).

FIG. 8A is a graph showing the production of isoprene in E. coli BL21cells with no vector.

FIG. 8B is a graph showing the production of isoprene in E. coli BL21cells with pCL-lac-Kudzu

FIG. 8C is a graph showing the production of isoprene in E. coli BL21cells with pTrcKudzu.

FIG. 8D is a graph showing the production of isoprene in E. coli BL21cells with pETN-HisKudzu.

FIG. 9A is a graph showing OD over time of fermentation of E. coliBL21/pTrcKudzu in a 14 liter fed batch fermentation.

FIG. 9B is a graph showing isoprene production over time of fermentationof E. coli BL21/pTrcKudzu in a 14 liter fed batch fermentation.

FIG. 10A is a graph showing the production of isoprene in Panteoacitrea. Control cells without recombinant kudzu isoprene synthase. Greydiamonds represent isoprene synthesis, black squares represent OD₆₀₀.

FIG. 10B is a graph showing the production of isoprene in Panteoa citreaexpressing pCL-lac Kudzu. Grey diamonds represent isoprene synthesis,black squares represent OD₆₀₀.

FIG. 10C is a graph showing the production of isoprene in Panteoa citreaexpressing pTrcKudzu. Grey diamonds represent isoprene synthesis, blacksquares represent OD₆₀₀.

FIG. 11 is a graph showing the production of isoprene in Bacillussubtilis expressing recombinant isoprene synthase. BG3594comK is a B.subtilis strain without plasmid (native isoprene production).CF443-BG3594comK is a B. subtilis strain with pBSKudzu (recombinantisoprene production). IS on the y-axis indicates isoprene.

FIG. 12 is the nucleotide sequence of pBS Kudzu #2 (SEQ ID NO:57).

FIG. 13 is the nucleotide sequence of kudzu isoprene synthasecodon-optimized for expression in Yarrowia (SEQ ID NO:8).

FIG. 14 is a map of pTrex3g comprising a kudzu isoprene synthase genecodon-optimized for expression in Yarrowia.

FIG. 15 is the nucleotide sequence of vector pSPZ1(MAP29Spb) (SEQ IDNO:11).

FIG. 16 is the nucleotide sequence of the synthetic kudzu (Puerariamontana) isoprene gene codon-optimized for expression in Yarrowia (SEQID NO:12).

FIG. 17 is the nucleotide sequence of the synthetic hybrid poplar(Populus alba×Populus tremula) isoprene synthase gene (SEQ ID NO:13).The ATG start codon is in bold and the stop codon is underlined.

FIG. 18A shows a schematic outlining construction of vectors pYLA 1,pYL1 and pYL2.

FIG. 18B shows a schematic outlining construction of the vectorpYLA(POP1).

FIG. 18C shows a schematic outlining construction of the vectorpYLA(KZ1)

FIG. 18D shows a schematic outlining construction of the vectorpYLI(KZ1)

FIG. 18E shows a schematic outlining construction of the vectorpYLI(MAP29)

FIG. 18F shows a schematic outlining construction of the vectorpYLA(MAP29)

FIG. 19A shows the MVA and DXP metabolic pathways for isoprene (based onF. Bouvier et al., Progress in Lipid Res. 44: 357-429, 2005). Thefollowing description includes alternative names for each polypeptide inthe pathways and a reference that discloses an assay for measuring theactivity of the indicated polypeptide (each of these references are eachhereby incorporated by reference in their entireties, particularly withrespect to assays for polypeptide activity for polypeptides in the MVAand DXP pathways). Mevalonate Pathway: AACT; Acetyl-CoAacetyltransferase, MvaE, EC 2.3.1.9. Assay: J. Bacteriol., 184:2116-2122, 2002; HMGS; Hydroxymethylglutaryl-CoA synthase, MvaS, EC2.3.3.10. Assay: J. Bacteriol., 184: 4065-4070, 2002; HMGR;3-Hydroxy-3-methylglutaryl-CoA reductase, MvaE, EC 1.1.1.34. Assay: J.Bacteriol., 184: 2116-2122, 2002; MVK; Mevalonate kinase, ERG12, EC2.7.1.36. Assay: Curr Genet. 19:9-14, 1991. PMK; Phosphomevalonatekinase, ERGS, EC 2.7.4.2, Assay: Mol Cell Biol., 11:620-631, 1991;DPMDC; Diphosphomevalonate decarboxylase, MVD1, EC 4.1.1.33. Assay:Biochemistry, 33:13355-13362, 1994; IDI; Isopentenyl-diphosphatedelta-isomerase, IDI1, EC 5.3.3.2. Assay: J. Biol. Chem.264:19169-19175, 1989. DXP Pathway: DXS; 1-Deoxyxylulose-5-phosphatesynthase, dxs, EC 2.2.1.7. Assay: PNAS, 94:12857-62, 1997; DXR;1-Deoxy-D-xylulose 5-phosphate reductoisomerase, dxr, EC 2.2.1.7. Assay:Eur. J. Biochem. 269:4446-4457, 2002; MCT;4-Diphosphocytidyl-2C-methyl-D-erythritol synthase, IspD, EC 2.7.7.60.Assay: PNAS, 97: 6451-6456, 2000; CMK;4-Diphosphocytidyl-2-C-methyl-D-erythritol kinase, IspE, EC 2.7.1.148.Assay: PNAS, 97:1062-1067, 2000; MCS; 2C-Methyl-D-erythritol2,4-cyclodiphosphate synthase, IspF, EC 4.6.1.12. Assay: PNAS,96:11758-11763, 1999; HDS; 1-Hydroxy-2-methyl-2-(E)-butenyl4-diphosphate synthase, ispG, EC 1.17.4.3. Assay: J. Org. Chem.,70:9168-9174, 2005; HDR; 1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphatereductase, IspH, EC 1.17.1.2. Assay: JACS, 126:12847-12855, 2004.

FIG. 19B illustrates the classical and modified MVA pathways. 1,acetyl-CoA acetyltransferase (AACT); 2, HMG-CoA synthase (HMGS); 3,HMG-CoA reductase (HMGR); 4, mevalonate kinase (MVK); 5,phosphomevalonate kinase (PMK); 6, diphosphomevalonate decarboxylase(MVD or DPMDC); 7, isopentenyl diphosphate isomerase (IDI); 8,phosphomevalonate decarboxylase (PMDC); 9, isopentenyl phosphate kinase(IPK). The classical MVA pathway proceeds from reaction 1 throughreaction 7 via reactions 5 and 6, while a modified MVA pathway goesthrough reactions 8 and 9. P and PP in the structural formula arephosphate and pyrophosphate, respectively. This figure was taken fromKoga and Morii, Microbiology and Mol. Biology Reviews, 71:97-120, 2007,which is incorporated by reference in its entirety, particularly withrespect to nucleic acids and polypeptides of the modified MVA pathway.The modified MVA pathway is present, for example, in some archaealorganisms, such as Methanosarcina mazei.

FIG. 20 shows graphs representing results of the GC-MS analysis ofisoprene production by recombinant Y. lipolytica strains without (left)or with (right) a kudzu isoprene synthase gene. The arrows indicate theelution time of the authentic isoprene standard.

FIG. 21 is a map of pTrcKudzu yIDI DXS Kan.

FIG. 22 is the nucleotide sequence of pTrcKudzu yIDI DXS Kan (SEQ IDNO:20).

FIG. 23A is a graph showing production of isoprene from glucose inBL21/pTrcKudzukan. Time 0 is the time of induction with IPTG (400 μmol).The x-axis is time after induction; the y-axis is OD₆₀₀ and the y2-axisis total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23B is a graph showing production of isoprene from glucose inBL21/pTrcKudzu yIDI kan. Time 0 is the time of induction with IPTG (400μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and they2-axis is total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23C is a graph showing production of isoprene from glucose inBL21/pTrcKudzu DXS kan. Time 0 is the time of induction with IPTG (400μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and they2-axis is total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23D is a graph showing production of isoprene from glucose inBL21/pTrcKudzu yIDI DXS kan. Time 0 is the time of induction with IPTG(400 μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ andthe y2-axis is total productivity of isoprene (μg/L headspace orspecific productivity (μg/L headspace/OD). Diamonds represent OD₆₀₀,circles represent total isoprene productivity (μg/L) and squaresrepresent specific productivity of isoprene (μg/L/OD).

FIG. 23E is a graph showing production of isoprene from glucose inBL21/pCL PtrcKudzu. Time 0 is the time of induction with IPTG (400μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and they2-axis is total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23F is a graph showing production of isoprene from glucose inBL21/pCL PtrcKudzu yIDI. Time 0 is the time of induction with IPTG (400μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and they2-axis is total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23G is a graph showing production of isoprene from glucose inBL21/pCL PtrcKudzu DXS. Time 0 is the time of induction with IPTG (400μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and they2-axis is total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23H is a graph showing production of isoprene from glucose inBL21/pTrcKudzuIDIDXSkan. The arrow indicates the time of induction withIPTG (400 μmol). The x-axis is time after induction; the y-axis is OD₆₀₀and the y2-axis is total productivity of isoprene (μg/L headspace orspecific productivity (μg/L headspace/OD). Black diamonds representOD₆₀₀, black triangles represent isoprene productivity (μg/L) and whitesquares represent specific productivity of isoprene (μg/L/OD).

FIG. 24 is a map of pTrcKKDyIkIS kan.

FIG. 25 is a nucleotide sequence of pTrcKKDyIkIS kan (SEQ ID NO:33).

FIG. 26 is a map of pCL PtrcUpperPathway.

FIG. 27 is a nucleotide sequence of pCL PtrcUpperPathway (SEQ ID NO:46).

FIG. 28 shows a map of the cassette containing the lower MVA pathway andyeast idi for integration into the B. subtilis chromosome at the nprElocus. nprE upstream/downstream indicates 1 kb each of sequence from thenprE locus for integration. aprE promoter (alkaline serine proteasepromoter) indicates the promoter (−35, −10, +1 transcription start site,RBS) of the aprE gene. MVK1 indicates the yeast mevalonate kinase gene.RBS-PMK indicates the yeast phosphomevalonte kinase gene with a BacillusRBS upstream of the start site. RBS-MPD indicates the yeastdiphosphomevalonate decarboxylase gene with a Bacillus RBS upstream ofthe start site. RBS-IDI indicates the yeast idi gene with a Bacillus RBSupstream of the start site. Terminator indicates the terminator alkalineserine protease transcription terminator from B. amyliquefaciens. SpecRindicates the spectinomycin resistance marker. “nprE upstream repeat foramp.” indicates a direct repeat of the upstream region used foramplification.

FIG. 29 is a nucleotide sequence of cassette containing the lower MVApathway and yeast idi for integration into the B. subtilis chromosome atthe nprE locus (SEQ ID NO:47).

FIG. 30 is a map of p9796-poplar.

FIG. 31 is a nucleotide sequence of p9796-poplar (SEQ ID NO:48).

FIG. 32 is a map of pTrcPoplar.

FIG. 33 is a nucleotide sequence of pTrcPoplar (SEQ ID NO:49).

FIG. 34 is a map of pTrcKudzu yIDI Kan.

FIG. 35 is a nucleotide sequence of pTrcKudzu yIDI Kan (SEQ ID NO:50).

FIG. 36 is a map of pTrcKudzuDXS Kan.

FIG. 37 is a nucleotide sequence of pTrcKudzuDXS Kan (SEQ ID NO:51).

FIG. 38 is a map of pCL PtrcKudzu.

FIG. 39 is a nucleotide sequence of pCL PtrcKudzu (SEQ ID NO:52).

FIG. 40 is a map of pCL PtrcKudzu A3.

FIG. 41 is a nucleotide sequence of pCL PtrcKudzu A3 (SEQ ID NO:53).

FIG. 42 is a map of pCL PtrcKudzu yIDI.

FIG. 43 is a nucleotide sequence of pCL PtrcKudzu yIDI (SEQ ID NO:54).

FIG. 44 is a map of pCL PtrcKudzu DXS.

FIG. 45 is a nucleotide sequence of pCL PtrcKudzu DXS (SEQ ID NO:55).

FIG. 46 shows graphs representing isoprene production from biomassfeedstocks. Panel A shows isoprene production from corn stover, Panel Bshows isoprene production from bagasse, Panel C shows isopreneproduction from softwood pulp, Panel D shows isoprene production fromglucose, and Panel E shows isoprene production from cells with noadditional feedstock. Grey squares represent OD₆₀₀ measurements of thecultures at the indicated times post-inoculation and black trianglesrepresent isoprene production at the indicated times post-inoculation.

FIG. 47A shows a graph representing isoprene production by BL21 (XDE3)pTrcKudzu yIDI DXS (kan) in a culture with no glucose added. Squaresrepresent OD₆₀₀, and triangles represent isoprene produced (μg/ml).

FIG. 47B shows a graph representing isoprene production from 1% glucosefeedstock invert sugar by BL21 (XDE3) pTrcKudzu yIDI DXS (kan). Squaresrepresent OD₆₀₀, and triangles represent isoprene produced (μg/ml).

FIG. 47C shows a graph representing isoprene production from 1% invertsugar feedstock by BL21 (XDE3) pTrcKudzu yIDI DXS (kan). Squaresrepresent OD₆₀₀, and triangles represent isoprene produced (μg/ml).

FIG. 47D shows a graph representing isoprene production from 1% AFEXcorn stover feedstock by BL21 (XDE3) pTrcKudzu yIDI DXS (kan). Squaresrepresent OD₆₀₀, and triangles represent isoprene produced (μg/ml).

FIG. 48 shows graphs demonstrating the effect of yeast extract ofisoprene production. Panel A shows the time course of optical densitywithin fermentors fed with varying amounts of yeast extract. Panel Bshows the time course of isoprene titer within fermentors fed withvarying amounts of yeast extract. The titer is defined as the amount ofisoprene produced per liter of fermentation broth. Panel C shows theeffect of yeast extract on isoprene production in E. coli grown infed-batch culture.

FIG. 49 shows graphs demonstrating isoprene production from a 500 Lbioreactor with E. coli cells containing the pTrcKudzu and yIDI and DXSplasmid. Panel A shows the time course of optical density within the500-L bioreactor fed with glucose and yeast extract. Panel B shows thetime course of isoprene titer within the 500-L bioreactor fed withglucose and yeast extract. The titer is defined as the amount ofisoprene produced per liter of fermentation broth. Panel C shows thetime course of total isoprene produced from the 500-L bioreactor fedwith glucose and yeast extract.

FIG. 50 is a map of pJMupperpathway2.

FIG. 51 is the nucleotide sequence of pJMupperpathway2 (SEQ ID NO:56).

FIG. 52 is a map of pBS Kudzu #2.

FIG. 53A is a graph showing growth during fermentation time of Bacillusexpressing recombinant kudzu isoprene synthase in 14 liter fed batchfermentation. Black diamonds represent a control strain (BG3594comK)without recombinant isoprene synthase (native isoprene production) andgrey triangles represent Bacillus with pBSKudzu (recombinant isopreneproduction).

FIG. 53B is a graph showing isoprene production during fermentation timeof Bacillus expressing recombinant kudzu isoprene synthase in 14 literfed batch fermentation. Black diamonds represent a control strain(BG3594comK) without recombinant isoprene synthase (native isopreneproduction) and grey triangles represent Bacillus with pBSKudzu(recombinant isoprene production).

FIG. 54 is a map of plasmid pET24 P. alba HGS.

FIGS. 55A and 55B are the nucleotide sequence of plasmid pET24 P. albaHGS (SEQ ID NO:87).

FIG. 56 is a schematic diagram showing restriction sites used forendonuclease digestion to construct plasmid EWL230 and compatiblecohesive ends between BspHI and NcoI sites.

FIG. 57 is a map of plasmid EWL230.

FIGS. 58A and 58B are the nucleotide sequence of plasmid EWL230 (SEQ IDNO:88).

FIG. 59 is a schematic diagram showing restriction sites used forendonuclease digestion to construct plasmid EWL244 and compatiblecohesive ends between NsiI and PstI sites.

FIG. 60 is a map of EWL244.

FIGS. 61A and 61B are the nucleotide sequence of plasmid EWL244 (SEQ IDNO:89).

FIG. 62 is a map of plasmids MCM484-487.

FIGS. 63A-63C are the nucleotide sequence of plasmid MCM484 (SEQ IDNO:90).

FIGS. 64A-64C are the nucleotide sequence of plasmid MCM485 (SEQ IDNO:91).

FIGS. 65A-65C are the nucleotide sequence of plasmid MCM486 (SEQ IDNO:92).

FIGS. 66A-66C are the nucleotide sequence of plasmid MCM487 (SEQ IDNO:93).

FIGS. 67A-67D are graphs of isoprene production by E. coli strain(EWL256) expressing genes from the MVA pathway and grown in fed-batchculture at the 15-L scale without yeast extract feeding. FIG. 67A showsthe time course of optical density within the 15-L bioreactor fed withglucose. FIG. 67B shows the time course of isoprene titer within the15-L bioreactor fed with glucose. The titer is defined as the amount ofisoprene produced per liter of fermentation broth. FIG. 67C shows thetime course of total isoprene produced from the 15-L bioreactor fed withglucose. FIG. 67D shows the total carbon dioxide evolution rate (TCER),or metabolic activity profile, within the 15-L bioreactor fed withglucose.

FIGS. 68A-68E are graphs of isoprene production by E. coli strain(EWL256) expressing genes from the MVA pathway and grown in fed-batchculture at the 15-L scale with yeast extract feeding. FIG. 68A shows thetime course of optical density within the 15-L bioreactor fed withglucose. FIG. 68B shows the time course of isoprene titer within the15-L bioreactor fed with glucose. The titer is defined as the amount ofisoprene produced per liter of fermentation broth. FIG. 68C shows thetime course of total isoprene produced from the 15-L bioreactor fed withglucose. FIG. 68D shows the volumetric productivity within the 15-Lbioreactor fed with glucose. An average value of 1.1 g/L/hr wasmaintained for a 40-hour period (23-63 hours) with yeast extractfeeding. FIG. 68E shows the carbon dioxide evolution rate (CER), ormetabolic activity profile, within the 15-L bioreactor fed with glucose.

FIGS. 69A-69D shows production of isoprene from different carbon sourcesvia the MVA (pathway). FIG. 69A shows growth of E. coli EWL256, whichcontains both the MVA pathway and isoprene synthase, on either glucose,biomass hydrolysate, glycerol, or acetate as the only carbon source. Thedifferent carbon sources were added to a concentration of 1% in themedia. A negative control with no added carbon source was included.Growth was measured as optical density at 600 nM. FIG. 69B showsspecific productivity of isoprene from E. coli EWL256 containing boththe MVA pathway and isoprene synthase when grown on either glucose,biomass hydrolysate, glycerol, or acetate as only carbon source. Thedifferent carbon sources were added to a concentration of 1% in themedia. A negative control with no added carbon source was included.Samples were taken 190 minutes, 255 minutes and 317 minutes afterinoculation and isoprene produced by the bacteria was measured usingGC-MS. FIG. 69C shows growth of E. coli EWL256 on either glucose orxylose as the only carbon source. The different carbon sources wereadded to a concentration of 1% in the media. A negative control with noadded carbon source was included. Growth was measured as optical densityat 600 nM. FIG. 69D shows specific productivity of isoprene from E. coliEWL256 when grown on either glucose or xylose as only carbon source. Thecarbon sources were added to a concentration of 1% in the media. Anegative control with no added carbon source was included. Samples weretaken 260 minutes, 322 minutes and 383 minutes after inoculation andisoprene produced by the bacteria was measured using GC-MS.

FIGS. 70A and 70B show the production of isoprene by E. coli strainsfrom glucose and from fatty acid, respectively. For FIG. 70A, elevencolonies from the transformation of WW4 with pMCM118, the plasmidbearing the lower mevalonic acid pathway, were picked to verify thepresence of the lower pathway. Cell from the colonies were cultured inTM3 medium containing 0.1% yeast extract and 2% glucose. Aliquots ofinduced culture were assayed for isoprene production after 4 hours ofinduction. All colonies showed the production of isoprene. The inducerIPTG had a strong growth inhibitory effect as was evident from the 3 to4.6-fold reduced cell density in going from 50 to 900 uM concentrationof the inducer (data not shown). The graph shows that higher induction,yields a higher specific titer of isoprene. For FIG. 70B, the productionculture was inoculated from a washed overnight culture at 1 to 10dilution. The culture was grown for several hours and induced with 50 uMIPTG. The left bar shows isoprene assay results four hours afterinduction followed by a one hour isoprene accumulation assay. The middlebar shows the one hour normalized value for the same culture with thesame induction period but analyzed by a 12 hour isoprene accumulationassay. The right bar shows the value for a one hour isopreneaccumulation assay of the culture that was induced for 13 hours.

FIG. 71 is a map of the E. coli-Streptomyces shuttle vector pUWL201PW(6400 bp) used for cloning isoprene synthase from Kudzu. Tsr,thiostrepton resistance gene. Picture is taken from Doumith et al., Mol.Gen. Genet. 264: 477-485, 2000.

FIG. 72 shows isoprene formation by Streptomyces albus wild type strain(“wt”) and strains harboring plasmid pUWL201PW (negative control) orpUWL201_iso (encoding isoprene synthase from Kudzu).

FIG. 73A is a map of the M. mazei archaeal Lower Pathway operon.

FIGS. 73B and 73C are the nucleotide sequence of the M. mazei archaeallower Pathway operon (SEQ ID NO:113).

FIG. 74A is a map of MCM376-MVK from M. mazei archaeal Lowerin pET200D.

FIGS. 74B and 74C are the nucleotide sequence of MCM376-MVK from M.mazei archaeal Lowerin pET200D (SEQ ID NO:114).

FIGS. 75A-75D show growth and specific productivity of isopreneproduction for EWL256 compared to RM11608-2. Growth (OD550) isrepresented by the white diamonds; specific productivity of isoprene isrepresented by the solid bars. The x-axis is time (hours) post-inductionwith either 200 (FIGS. 75A and 75B) or 400 (FIGS. 75C and 75D) uM IPTG.Y-1 axis is productivity of isoprene (ug/L/OD/hr) and Y-2 is arbitraryunits of optical density at a wavelength of 550. These values for theOD550 must be multiplied by 6.66 to obtain the actual OD of the culture.

FIG. 76 is a map of plasmid pBBRCMPGI1.5-pgl.

FIGS. 77A and 77B are the nucleotide sequence of plasmidpBBRCMPGI1.5-pgl (SEQ ID NO:122).

FIGS. 78A-78F are graphs of isoprene production by E. coli strainexpressing M. mazei mevalonate kinase, P. alba isoprene synthase, andpgl (RHM111608-2), and grown in fed-batch culture at the 15-L scale.FIG. 78A shows the time course of optical density within the 15-Lbioreactor fed with glucose. FIG. 78B shows the time course of isoprenetiter within the 15-L bioreactor fed with glucose. The titer is definedas the amount of isoprene produced per liter of fermentation broth.Method for calculating isoprene: cumulative isoprene produced in 59 hrs,g/Fermentor volume at 59 hrs, L [=] g/L broth. FIG. 78C also shows thetime course of isoprene titer within the 15-L bioreactor fed withglucose. Method for calculating isoprene: ∫(Instantaneous isopreneproduction rate, g/L/hr)dt from t=0 to 59 hours [=] g/L broth. FIG. 78Dshows the time course of total isoprene produced from the 15-Lbioreactor fed with glucose. FIG. 78E shows volumetric productivitywithin the 15-L bioreactor fed with glucose. FIG. 78F shows carbondioxide evolution rate (CER), or metabolic activity profile, within the15-L bioreactor fed with glucose.

FIG. 79A is a map of plasmid pJ201:19813.

FIGS. 79B and 79C are the nucleotide sequence of pJ201:19813 (SEQ IDNO:123).

DETAILED DESCRIPTION OF THE INVENTION

The invention features compositions and methods for the production ofincreased amounts of isoprene. In particular, these compositions andmethods increase the rate of isoprene production and increase the totalamount of isoprene that is produced. For example, cell culture systemsthat generate 4.8×10⁴ nmole/g_(wcm)/hr of isoprene have been produced(Table 1). The efficiency of these systems is demonstrated by theconversion of ˜23.6 molar % yield (10.7 weight % yield) of the carbonthat the cells consume from a cell culture medium into isoprene (%carbon yield). As shown in the Examples and Table 2, approximately 60.5g of isoprene per liter of broth was generated. Isoprene was produced ata peak specific rate of 1.88×10⁵ nmol/OD/hr (1.88×10⁵ nmole/g_(wcm)/hr).If desired, even greater amounts of isoprene can be obtained using otherconditions, such as those described herein. In some embodiments, arenewable carbon source is used for the production of isoprene. Thecompositions and methods of the present invention are desirable becausethey allow high isoprene yield per cell, high carbon yield, highisoprene purity, high productivity, low energy usage, low productioncost and investment, and minimal side reactions. This efficient, largescale, biosynthetic process for isoprene production provides an isoprenesource for synthetic isoprene-based rubber and provides a desirable,low-cost alternative to using natural rubber.

As discussed further below, the amount of isoprene produced by cells canbe greatly increased by introducing a heterologous nucleic acid encodingan isoprene synthase polypeptide (e.g., a plant isoprene synthasepolypeptide) into the cells. Isoprene synthase polypeptides convertdimethylallyl diphosphate (DMAPP) into isoprene. As shown in theExamples, a heterologous Pueraria Montana (kudzu) or Populus alba(Poplar) isoprene synthase polypeptide was expressed in a variety ofhost cells, such as Escherichia coli, Panteoa citrea, Bacillus subtilis,Yarrowia lipolytica, and Trichoderma reesei. As also shown in theExamples, a heterologous Methanosarcina mazei (M. mazei) mevalonatekinase (MVK) was expressed in host cells such as Escherichia coli toincrease isoprene production. All of these cells produced more isoprenethan the corresponding cells without the heterologous isoprene synthasepolypeptide. As illustrated in Tables 1 and 2, large amounts of isopreneare produced using the methods described herein. For example, B.subtilis cells with a heterologous isoprene synthase nucleic acidproduced approximately 10-fold more isoprene in a 14 liter fermentorthan the corresponding control B. subtilis cells without theheterologous nucleic acid (Table 2). The production of 60.5 g ofisoprene per liter of broth (mg/L, wherein the volume of broth includesboth the volume of the cell medium and the volume of the cells) by E.coli and 30 mg/L by B. subtilis in fermentors indicates that significantamounts of isoprene can be generated (Table 2). If desired, isoprene canbe produced on an even larger scale or other conditions described hereincan be used to further increase the amount of isoprene. The vectorslisted in Tables 1 and 2 and the experimental conditions are describedin further detail below and in the Examples section.

TABLE 1 Exemplary yields of isoprene from a shake flask using the cellcultures and methods of the invention. The assay for measuring isopreneproduction is described in Example I, part II. For this assay, a samplewas removed at one or more time points from the shake flask and culturedfor 30 minutes. The amount of isoprene produced in this sample was thenmeasured. The headspace concentration and specific rate of isopreneproduction are listed in Table 1 and described further herein. IsopreneProduction in a Headspace vial* Headspace Specific Rate concentrationμg/L_(broth)/hr/OD Strain μg/L_(gas) (nmol/g_(wcm)/hr) E. coliBL21/pTrcKudzu IS 1.40 53.2 (781.2)  E. coli BL21/Pcl DXS yidi 7.61289.1  Kudzu IS (4.25 × 10³) E. coli BL21/MCM127 with 23.0 874.1  kudzuIS and entire MVA (1.28 × 10⁴) pathway E. coli BL21/Pet N- 1.49 56.6HisKudzu IS (831.1)  Pantoea citrea/pTrcKudzu 0.66 25.1 IS (368.6)  E.coli w/Poplar IS —  5.6 [Miller (2001)] (82.2) Bacillis licheniformisFall —  4.2 U.S. Pat. No. 5,849,970 (61.4) Yarrowia lipolytica with~0.05 μg/L ~2   kudzu isoprene synthase (~30)   Trichoderma reesei with~0.05 μg/L ~2   kudzu isoprene synthase (~30)   E. coli BL21/ 85.9 3.2 ×10³ pTrcKKD_(y)I_(k)IS with kudzu (4.8 × 10⁴) IS and lower MVA pathway*Normalized to 1 mL of 1 OD₆₀₀, cultured for 1 hour in a sealedheadspace vial with a liquid to headspace volume ratio of 1:19.

TABLE 2 Exemplary yields of isoprene in a fermentor using the cellcultures and methods of the invention. The assay for measuring isopreneproduction is described in Example I, part II. For this assay, a sampleof the off- gas of the fermentor was taken and analyzed for the amountof isoprene. The peak headspace concentration (which is the highestheadspace concentration during the fermentation), titer (which is thecumulative, total amount of isoprene produced per liter of broth), andpeak specific rate of isoprene production (which is the highest specificrate during the fermentation) are listed in Table 2 and describedfurther herein. Isoprene Production in Fermentors Peak Headspace PeakSpecific rate concentration** Titer μg/L_(broth)/hr/OD Strain(ug/L_(gas)) (mg/L_(broth)) (nmol/g_(wcm)/hr) E. coli BL21/ 52 41.2 37 pTrcKudzu with (543.3) Kudzu IS E. coli 3 3.5  21.4 FM5/pTrcKudzu IS(308.1) E. coli BL21/triple 285 300 240   strain (DXS, yidi, (3.52 ×10³) IS) E. coli FM5/triple 50.8 29 180.8 strain (DXS, yidi, (2.65 ×10³) IS) E. coli/MCM127 1094 250 875   with Kudzu IS and (1.28 × 10⁴)entire MVA pathway Bacillus subtilis 1.5 2.5  0.8 wild-type  (11.7)Bacillus pBS Kudzu 16.6 ~30 (over 5 IS 100 hours)  (73.4) BacillusMarburg 2.04 0.61  24.5 6051 [Wagner and (359.8) Fall (1999)] BacillusMarburg 0.7 0.15  6.8 6051 Fall U.S. (100)   Pat. No. 5,849,970 E. coli2.03 × 10⁴ 3.22 × 10⁴  5.9 × 10³ BL21/pCLPtrcUpper (8.66 × 10⁴) Pathwayand gil.2KKDyI and pTrcAlba-mMVK E. coli 3.22 × 10⁴ 6.05 × 10⁴ 1.28 ×10⁴ BL21/pCLPtrcUpper (1.88 × 10⁵) Pathway and gi1.2KKDyI andpTrcAlba-mMVK plus pBBRCMPGI1.5pgl **Normalized to an off-gas flow rateof 1 vvm (1 volume off-gas per 1 L_(broth) per minute).

Additionally, isoprene production by cells that contain a heterologousisoprene synthase nucleic acid can be enhanced by increasing the amountof a 1-deoxy-D-xylulose-5-phosphate synthase (DXS) polypeptide and/or anisopentenyl diphosphate isomerase (IDI) polypeptide expressed by thecells. For example, a DXS nucleic acid and/or an IDI nucleic acid can beintroduced into the cells. The DXS nucleic acid may be a heterologousnucleic acid or a duplicate copy of an endogenous nucleic acid.Similarly, the IDI nucleic acid may be a heterologous nucleic acid or aduplicate copy of an endogenous nucleic acid. In some embodiments, theamount of DXS and/or IDI polypeptide is increased by replacing theendogenous DXS and/or IDI promoters or regulatory regions with otherpromoters and/or regulatory regions that result in greater transcriptionof the DXS and/or IDI nucleic acids. In some embodiments, the cellscontain both a heterologous nucleic acid encoding an isoprene synthasepolypeptide (e.g., a plant isoprene synthase nucleic acid) and aduplicate copy of an endogenous nucleic acid encoding an isoprenesynthase polypeptide.

The encoded DXS and IDI polypeptides are part of the DXP pathway for thebiosynthesis of isoprene (FIG. 19A). DXS polypeptides convert pyruvateand D-glyceraldehyde-3-phosphate into 1-deoxy-D-xylulose-5-phosphate.While not intending to be bound by any particular theory, it is believedthat increasing the amount of DXS polypeptide increases the flow ofcarbon through the DXP pathway, leading to greater isoprene production.IDI polypeptides catalyze the interconversion of isopentenyl diphosphate(IPP) and dimethylallyl diphosphate (DMAPP). While not intending to bebound by any particular theory, it is believed that increasing theamount of IDI polypeptide in cells increases the amount of IPP that isconverted into DMAPP, which in turn is converted into isoprene.

For example, fermentation of E. coli cells with a kudzu isoprenesynthase, S. cerevisia IDI, and E. coli DXS nucleic acids was used toproduce isoprene. The levels of isoprene varied from 50 to 300 μg/L overa time period of 15 hours (Example 7, part VII). As another example,fermentation of E. coli with M. mazei mevalonate kinase (MVK), P. albaisoprene synthase, the upper MVA pathway, and the integrated lower MVApathway was used to produce isoprene. The levels of isoprene varied from32 to 35.6 g/L over a time period of 67 hours (Example 10, part III).

In yet another example, fermentation of E. coli with M. mazei mevalonatekinase (MVK), P. alba isoprene synthase, pgl over-expression(RHM111608-2), the upper MVA pathway, and the integrated lower MVApathway were used to produce isoprene. The levels of isoprene vary from33.2 g/L to 40.0 g/L over a time period of 40 hours or 48.6 g/L to 60.5g/L over a time period of 59 hours (Example 13, part (ii)).

In some embodiments, the presence of heterologous or extra endogenousisoprene synthase, IDI, and DXS nucleic acids causes cells to grow morereproducibly or remain viable for longer compared to the correspondingcell with only one or two of these heterologous or extra endogenousnucleic acids. For example, cells containing heterologous isoprenesynthase, IDI, and DXS nucleic acids grew better than cells with onlyheterologous isoprene synthase and DXS nucleic acids or with only aheterologous isoprene synthase nucleic acid. Also, heterologous isoprenesynthase, IDI, and DXS nucleic acids were successfully operably linkedto a strong promoter on a high copy plasmid that was maintained by E.coli cells, suggesting that large amounts of these polypeptides could beexpressed in the cells without causing an excessive amount of toxicityto the cells. While not intending to be bound to a particular theory, itis believed that the presence of heterologous or extra endogenousisoprene synthase and IDI nucleic acids may reduce the amount of one ormore potentially toxic intermediates that would otherwise accumulate ifonly a heterologous or extra endogenous DXS nucleic acid was present inthe cells.

In some embodiments, the production of isoprene by cells that contain aheterologous isoprene synthase nucleic acid is augmented by increasingthe amount of a MVA polypeptide expressed by the cells (FIGS. 19A and19B). Exemplary MVA pathways polypeptides include any of the followingpolypeptides: acetyl-CoA acetyltransferase (AA-CoA thiolase)polypeptides, 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase)polypeptides, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoAreductase) polypeptides, mevalonate kinase (MVK) polypeptides,phosphomevalonate kinase (PMK) polypeptides, diphosphomevalontedecarboxylase (MVD) polypeptides, phosphomevalonate decarboxylase (PMDC)polypeptides, isopentenyl phosphate kinase (IPK) polypeptides, IDIpolypeptides, and polypeptides (e.g., fusion polypeptides) having anactivity of two or more MVA pathway polypeptides. For example, one ormore MVA pathway nucleic acids can be introduced into the cells. In someembodiments, the cells contain the upper MVA pathway, which includesAA-CoA thiolase, HMG-CoA synthase, and HMG-CoA reductase nucleic acids.In some embodiments, the cells contain the lower MVA pathway, whichincludes MVK, PMK, MVD, and IDI nucleic acids. In some embodiments, thecells contain the entire MVA pathway, which includes AA-CoA thiolase,HMG-CoA synthase, HMG-CoA reductase, MVK, PMK, MVD, and IDI nucleicacids. In some embodiments, the cells contain an entire MVA pathway thatincludes AA-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, MVK,PMDC, IPK, and IDI nucleic acids. The MVA pathway nucleic acids may beheterologous nucleic acids or duplicate copies of endogenous nucleicacids. In some embodiments, the amount of one or more MVA pathwaypolypeptides is increased by replacing the endogenous promoters orregulatory regions for the MVA pathway nucleic acids with otherpromoters and/or regulatory regions that result in greater transcriptionof the MVA pathway nucleic acids. In some embodiments, the cells containboth a heterologous nucleic acid encoding an isoprene synthasepolypeptide (e.g., a plant isoprene synthase nucleic acid) and aduplicate copy of an endogenous nucleic acid encoding an isoprenesynthase polypeptide.

For example, E. coli cells containing a nucleic acid encoding a kudzuisoprene synthase polypeptide and nucleic acids encoding Saccharomycescerevisia MVK, PMK, MVD, and IDI polypeptides generated isoprene at arate of 6.67×10⁴ nmol/L_(broth)/OD₆₀₀/hr (see Example 8). Additionally,a 14 liter fermentation of E. coli cells with nucleic acids encodingEnterococcus faecalis AA-CoA thiolase, HMG-CoA synthase, and HMG-CoAreductase polypeptides produced 22 grams of mevalonic acid (anintermediate of the MVA pathway). A shake flask of these cells produced2-4 grams of mevalonic acid per liter. These results indicate thatheterologous MVA pathways nucleic acids are active in E. coli. E. colicells that contain nucleic acids for both the upper MVA pathway and thelower MVA pathway as well as a kudzu isoprene synthase (strain MCM 127)produced significantly more isoprene (874

g/L_(broth)/hr/OD) compared to E. coli cells with nucleic acids for onlythe lower MVA pathway and the kudzu isoprene synthase (strain MCM 131)(see Table 3 and Example 8, part VIII).

As another example, E. coli cells containing a nucleic acid encoding aP. alba isoprene synthase polypeptide and a nucleic acid encoding M.mazei MVK polypeptide generated 320.6 g (at a peak specific rate of9.54×10⁴ nmol/L_(broth)/OD₆₀₀/hr (i.e. 9.5×10⁻⁵ mol/L_(broth)/OD₆₀₀/hr))of isoprene during a 67 hour fermentation in the absence of yeastextract feeding or 395.5 g (at a peak specific rate of 8.66×10⁴nmol/L_(broth)/OD₆₀₀/hr) during a 68 hour fermentation in the presenceof yeast extract feeding (see Example 10).

In some embodiments, at least a portion of the cells maintain theheterologous isoprene synthase, DXS, IDI, and/or MVA pathway nucleicacid for at least about 5, 10, 20, 50, 75, 100, 200, 300, or more celldivisions in a continuous culture (such as a continuous culture withoutdilution). In some embodiments of any of the aspects of the invention,the nucleic acid comprising the heterologous or duplicate copy of anendogenous isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acidalso comprises a selective marker, such as a kanamycin, ampicillin,carbenicillin, gentamicin, hygromycin, phleomycin, bleomycin, neomycin,or chloramphenicol antibiotic resistance nucleic acid.

As indicated in Example 7, part VI, the amount of isoprene produced canbe further increased by adding yeast extract to the cell culture mediumusing E. coli cells with kudzu isoprene synthase, S. cerevisia IDI, andE. coli DXS nucleic acids to produce isoprene. In particular, the amountof isoprene produced was linearly proportional to the amount of yeastextract in the cell medium for the concentrations tested (FIG. 48C).Additionally, approximately 0.11 grams of isoprene per liter of brothwas produced from a cell medium with yeast extract and glucose (Example7, part VIII). Increasing the amount of yeast extract in the presence ofglucose resulted in more isoprene being produced than increasing theamount of glucose in the presence of yeast extract. Also, increasing theamount of yeast extract allowed the cells to produce a high level ofisoprene for a longer length of time and improved the health of thecells.

Isoprene production was also demonstrated using three types ofhydrolyzed biomass (bagasse, corn stover, and soft wood pulp) as thecarbon source (FIGS. 46A-C and FIGS. 69A and 69B). E. coli cells withkudzu isoprene synthase, S. cerevisia IDI, and E. coli DXS nucleic acidsproduced as much isoprene from these hydrolyzed biomass carbon sourcesas from the equivalent amount of glucose (e.g., 1% glucose, w/v). E.coli cells expressing P. alba isoprene synthase and the MVA pathwayproduced isoprene at a higher initial growth rate from ammonia fiberexpansion (AFEX) pretreated corn stover than from the equivalent amountof glucose. (FIGS. 69A and 69B). If desired, any other biomass carbonsource can be used in the compositions and methods of the invention.Biomass carbon sources are desirable because they are cheaper than manyconventional cell mediums, thereby facilitating the economicalproduction of isoprene.

Additionally, invert sugar was shown to function as a carbon source forthe generation of isoprene (FIG. 47D).

Additionally, xylose, acetate, and glycerol were also shown to functionas a carbon source for the generation of isoprene (FIGS. 69A-69D). Forexample, E. coli cells with P. alba isoprene synthase and the MVApathway grown on acetate as the only carbon source had a specificproductivity of isoprene about twice as high as during growth on glucose(Example 10, Part IV; FIGS. 69A and 69B).

In some embodiments, an oil is included in the cell medium. For example,B. subtilis cells containing a kudzu isoprene synthase nucleic acidproduced isoprene when cultured in a cell medium containing an oil and asource of glucose (Example 4, part III). As another example, E. colifadR atoC mutant cells containing the upper and lower MVA pathway pluskudzu isoprene synthase produced isoprene when cultured in a cell mediumcontaining palm oil and a source of glucose (Example 12, part II). Insome embodiments, more than one oil (such as 2, 3, 4, 5, or more oils)is included in the cell medium. While not intending to be bound to anyparticular theory, it is believed that (i) the oil may increase theamount of carbon in the cells that is available for conversion toisoprene, (ii) the oil may increase the amount of acetyl-CoA in thecells, thereby increasing the carbon flow through the MVA pathway,and/or (ii) the oil may provide extra nutrients to the cells, which isdesirable since a lot of the carbon in the cells is converted toisoprene rather than other products. In some embodiments, cells that arecultured in a cell medium containing oil naturally use the MVA pathwayto produce isoprene or are genetically modified to contain nucleic acidsfor the entire MVA pathway. In some embodiments, the oil is partially orcompletely hydrolyzed before being added to the cell culture medium tofacilitate the use of the oil by the host cells.

Exemplary Polypeptides and Nucleic Acids

Various isoprene synthase, DXS, IDI, and/or MVA pathway polypeptides andnucleic acids can be used in the compositions and methods of theinvention.

As used herein, “polypeptides” includes polypeptides, proteins,peptides, fragments of polypeptides, and fusion polypeptides thatinclude part or all of a first polypeptide (e.g., an isoprene synthase,DXS, IDI, or MVA pathway polypeptide) and part or all of a secondpolypeptide (e.g., a peptide that facilitates purification or detectionof the fusion polypeptide, such as a His-tag). In some embodiments, thefusion polypeptide has an activity of two or more MVA pathwaypolypeptides (such as AA-CoA thiolase and HMG-CoA reductasepolypeptides). In some embodiments, the polypeptide is anaturally-occurring polypeptide (such as the polypeptide encoded by anEnterococcus faecalis mvaE nucleic acid) that has an activity of two ormore MVA pathway polypeptides.

In various embodiments, a polypeptide has at least or about 50, 100,150, 175, 200, 250, 300, 350, 400, or more amino acids. In someembodiments, the polypeptide fragment contains at least or about 25, 50,75, 100, 150, 200, 300, or more contiguous amino acids from afull-length polypeptide and has at least or about 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of an activity of acorresponding full-length polypeptide. In particular embodiments, thepolypeptide includes a segment of or the entire amino acid sequence ofany naturally-occurring isoprene synthase, DXS, IDI, or MVA pathwaypolypeptide. In some embodiments, the polypeptide has one or moremutations compared to the sequence of a wild-type (i.e., a sequenceoccurring in nature) isoprene synthase, DXS, IDI, or MVA pathwaypolypeptide.

In some embodiments, the polypeptide is an isolated polypeptide. As usedherein, an “isolated polypeptide” is not part of a library ofpolypeptides, such as a library of 2, 5, 10, 20, 50 or more differentpolypeptides and is separated from at least one component with which itoccurs in nature. An isolated polypeptide can be obtained, for example,by expression of a recombinant nucleic acid encoding the polypeptide.

In some embodiments, the polypeptide is a heterologous polypeptide. By“heterologous polypeptide” is meant a polypeptide whose amino acidsequence is not identical to that of another polypeptide naturallyexpressed in the same host cell.

As used herein, a “nucleic acid” refers to two or moredeoxyribonucleotides and/or ribonucleotides in either single ordouble-stranded form. In some embodiments, the nucleic acid is arecombinant nucleic acid. By “recombinant nucleic acid” means a nucleicacid of interest that is free of one or more nucleic acids (e.g., genes)which, in the genome occurring in nature of the organism from which thenucleic acid of interest is derived, flank the nucleic acid of interest.The term therefore includes, for example, a recombinant DNA which isincorporated into a vector, into an autonomously replicating plasmid orvirus, or into the genomic DNA of a prokaryote or eukaryote, or whichexists as a separate molecule (e.g., a cDNA, a genomic DNA fragment, ora cDNA fragment produced by PCR or restriction endonuclease digestion)independent of other sequences. In some embodiments, an isoprenesynthase, DXS, IDI, or MVA pathway nucleic acid is operably linked toanother nucleic acid encoding all or a portion of another polypeptidesuch that the recombinant nucleic acid encodes a fusion polypeptide thatincludes an isoprene synthase, DXS, IDI, or MVA pathway polypeptide andall or part of another polypeptide (e.g., a peptide that facilitatespurification or detection of the fusion polypeptide, such as a His-tag).In some embodiments, part or all of a recombinant nucleic acid ischemically synthesized.

In some embodiments, the nucleic acid is a heterologous nucleic acid. By“heterologous nucleic acid” is meant a nucleic acid whose nucleic acidsequence is not identical to that of another nucleic acid naturallyfound in the same host cell.

In particular embodiments, the nucleic acid includes a segment of or theentire nucleic acid sequence of any naturally-occurring isoprenesynthase, DXS, IDI, or MVA pathway nucleic acid. In some embodiments,the nucleic acid includes at least or about 50, 100, 150, 200, 300, 400,500, 600, 700, 800, or more contiguous nucleotides from anaturally-occurring isoprene synthase nucleic acid DXS, IDI, or MVApathway nucleic acid. In some embodiments, the nucleic acid has one ormore mutations compared to the sequence of a wild-type (i.e., a sequenceoccurring in nature) isoprene synthase, DXS, IDI, or MVA pathway nucleicacid. In some embodiments, the nucleic acid has one or more mutations(e.g., a silent mutation) that increase the transcription or translationof isoprene synthase, DXS, IDI, or MVA pathway nucleic acid. In someembodiments, the nucleic acid is a degenerate variant of any nucleicacid encoding an isoprene synthase, DXS, IDI, or MVA pathwaypolypeptide.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without affecting the amino acidsequence of an encoded polypeptide. The skilled artisan is well aware ofthe “codon-bias” exhibited by a specific host cell in usage ofnucleotide codons to specify a given amino acid. Therefore, whensynthesizing a nucleic acid for improved expression in a host cell, itis desirable in some embodiments to design the nucleic acid such thatits frequency of codon usage approaches the frequency of preferred codonusage of the host cell.

The accession numbers of exemplary isoprene synthase, DXS, IDI, and/orMVA pathway polypeptides and nucleic acids are listed in Appendix 1 (theaccession numbers of Appendix 1 and their corresponding sequences areherein incorporated by reference in their entireties, particularly withrespect to the amino acid and nucleic acid sequences of isoprenesynthase, DXS, IDI, and/or MVA pathway polypeptides and nucleic acids).The Kegg database also contains the amino acid and nucleic acidsequences of numerous exemplary isoprene synthase, DXS, IDI, and/or MVApathway polypeptides and nucleic acids (see, for example, the world-wideweb at “genome.jp/kegg/pathway/map/map00100.html” and the sequencestherein, which are each hereby incorporated by reference in theirentireties, particularly with respect to the amino acid and nucleic acidsequences of isoprene synthase, DXS, IDI, and/or MVA pathwaypolypeptides and nucleic acids). In some embodiments, one or more of theisoprene synthase, DXS, IDI, and/or MVA pathway polypeptides and/ornucleic acids have a sequence identical to a sequence publicly availableon Dec. 12, 2007 or Dec. 11, 2008, such as any of the sequences thatcorrespond to any of the accession numbers in Appendix 1 or any of thesequences present in the Kegg database. Additional exemplary isoprenesynthase, DXS, IDI, and/or MVA pathway polypeptides and nucleic acidsare described further below.

Exemplary Isoprene Synthase Polypeptides and Nucleic Acids

As noted above, isoprene synthase polypeptides convert dimethylallyldiphosphate (DMAPP) into isoprene. Exemplary isoprene synthasepolypeptides include polypeptides, fragments of polypeptides, peptides,and fusions polypeptides that have at least one activity of an isoprenesynthase polypeptide. Standard methods can be used to determine whethera polypeptide has isoprene synthase polypeptide activity by measuringthe ability of the polypeptide to convert DMAPP into isoprene in vitro,in a cell extract, or in vivo. In an exemplary assay, cell extracts areprepared by growing a strain (e.g., the E. coli/pTrcKudzu straindescribed herein) in the shake flask method as described in Example 1.After induction is complete, approximately 10 mLs of cells are pelletedby centrifugation at 7000×g for 10 minutes and resuspended in 5 ml ofPEB without glycerol. The cells are lysed using a French Pressure cellusing standard procedures. Alternatively the cells are treated withlysozyme (Ready-Lyse lysozyme solution; EpiCentre) after a freeze/thawat −80 C.

Isoprene synthase polypeptide activity in the cell extract can bemeasured, for example, as described in Silver et al., J. Biol. Chem.270:13010-13016, 1995 and references therein, which are each herebyincorporated by reference in their entireties, particularly with respectto assays for isoprene synthase polypeptide activity. DMAPP (Sigma) isevaporated to dryness under a stream of nitrogen and rehydrated to aconcentration of 100 mM in 100 mM potassium phosphate buffer pH 8.2 andstored at −20° C. To perform the assay, a solution of 5 μl of 1M MgCl₂,1 mM (250

g/ml) DMAPP, 65

l of Plant Extract Buffer (PEB) (50 mM Tris-HCl, pH 8.0, 20 mM MgCl₂, 5%glycerol, and 2 mM DTT) is added to 25

l of cell extract in a 20 ml Headspace vial with a metal screw cap andteflon coated silicon septum (Agilent Technologies) and cultured at 37°C. for 15 minutes with shaking. The reaction is quenched by adding 200

l of 250 mM EDTA and quantified by GC/MS as described in Example 1, partII.

Exemplary isoprene synthase nucleic acids include nucleic acids thatencode a polypeptide, fragment of a polypeptide, peptide, or fusionpolypeptide that has at least one activity of an isoprene synthasepolypeptide. Exemplary isoprene synthase polypeptides and nucleic acidsinclude naturally-occurring polypeptides and nucleic acids from any ofthe source organisms described herein as well as mutant polypeptides andnucleic acids derived from any of the source organisms described herein.

In some embodiments, the isoprene synthase polypeptide or nucleic acidis from the family Fabaceae, such as the Faboideae subfamily. In someembodiments, the isoprene synthase polypeptide or nucleic acid is apolypeptide or nucleic acid from Pueraria montana (kudzu) (Sharkey etal., Plant Physiology 137: 700-712, 2005), Pueraria lobata, poplar (suchas Populus alba, Populus nigra, Populus trichocarpa, Populusalba×tremula (CAC35696), or Populus alba) (Sasaki et al., FEBS Letters579(11): 2514-2518, 2005; Miller et al., Planta 213: 483-487, 2001),aspen (such as Populus tremuloides) (Silver et al., JBC 270(22):13010-1316, 1995), or English Oak (Quercus robur) (Zimmer et al., WO98/02550), which are each hereby incorporated by reference in theirentireties, particularly with respect to isoprene synthase nucleic acidsand the expression of isoprene synthase polypeptides. Suitable isoprenesynthases include, but are not limited to, those identified by GenbankAccession Nos. AY341431, AY316691, AY279379, AJ457070, and AY182241,which are each hereby incorporated by reference in their entireties,particularly with respect to sequences of isoprene synthase nucleicacids and polypeptides. In some embodiments, the isoprene synthasepolypeptide or nucleic acid is not a naturally-occurring polypeptide ornucleic acid from Quercus robur (i.e., the isoprene synthase polypeptideor nucleic acid is an isoprene synthase polypeptide or nucleic acidother than a naturally-occurring polypeptide or nucleic acid fromQuercus robur). In some embodiments, the isoprene synthase nucleic acidor polypeptide is a naturally-occurring polypeptide or nucleic acid frompoplar. In some embodiments, the isoprene synthase nucleic acid orpolypeptide is not a naturally-occurring polypeptide or nucleic acidfrom poplar.

Exemplary DXS Polypeptides and Nucleic Acids

As noted above, 1-deoxy-D-xylulose-5-phosphate synthase (DXS)polypeptides convert pyruvate and D-glyceraldehyde-3-phosphate into1-deoxy-D-xylulose-5-phosphate. Exemplary DXS polypeptides includepolypeptides, fragments of polypeptides, peptides, and fusionspolypeptides that have at least one activity of a DXS polypeptide.Standard methods (such as those described herein) can be used todetermine whether a polypeptide has DXS polypeptide activity bymeasuring the ability of the polypeptide to convert pyruvate andD-glyceraldehyde-3-phosphate into 1-deoxy-D-xylulose-5-phosphate invitro, in a cell extract, or in vivo. Exemplary DXS nucleic acidsinclude nucleic acids that encode a polypeptide, fragment of apolypeptide, peptide, or fusion polypeptide that has at least oneactivity of a DXS polypeptide. Exemplary DXS polypeptides and nucleicacids include naturally-occurring polypeptides and nucleic acids fromany of the source organisms described herein as well as mutantpolypeptides and nucleic acids derived from any of the source organismsdescribed herein.

Exemplary IDI Polypeptides and Nucleic Acids

Isopentenyl diphosphate isomerase polypeptides (isopentenyl-diphosphatedelta-isomerase or IDI) catalyses the interconversion of isopentenyldiphosphate (IPP) and dimethylallyl diphosphate (DMAPP) (e.g.,converting IPP into DMAPP and/or converting DMAPP into IPP). ExemplaryIDI polypeptides include polypeptides, fragments of polypeptides,peptides, and fusions polypeptides that have at least one activity of anIDI polypeptide. Standard methods (such as those described herein) canbe used to determine whether a polypeptide has IDI polypeptide activityby measuring the ability of the polypeptide to interconvert IPP andDMAPP in vitro, in a cell extract, or in vivo. Exemplary IDI nucleicacids include nucleic acids that encode a polypeptide, fragment of apolypeptide, peptide, or fusion polypeptide that has at least oneactivity of an IDI polypeptide. Exemplary IDI polypeptides and nucleicacids include naturally-occurring polypeptides and nucleic acids fromany of the source organisms described herein as well as mutantpolypeptides and nucleic acids derived from any of the source organismsdescribed herein.

Exemplary MVA Pathway Polypeptides and Nucleic Acids

Exemplary MVA pathway polypeptides include acetyl-CoA acetyltransferase(AA-CoA thiolase) polypeptides, 3-hydroxy-3-methylglutaryl-CoA synthase(HMG-CoA synthase) polypeptides, 3-hydroxy-3-methylglutaryl-CoAreductase (HMG-CoA reductase) polypeptides, mevalonate kinase (MVK)polypeptides, phosphomevalonate kinase (PMK) polypeptides,diphosphomevalonte decarboxylase (MVD) polypeptides, phosphomevalonatedecarboxylase (PMDC) polypeptides, isopentenyl phosphate kinase (IPK)polypeptides, IDI polypeptides, and polypeptides (e.g., fusionpolypeptides) having an activity of two or more MVA pathwaypolypeptides. In particular, MVA pathway polypeptides includepolypeptides, fragments of polypeptides, peptides, and fusionspolypeptides that have at least one activity of an MVA pathwaypolypeptide. Exemplary MVA pathway nucleic acids include nucleic acidsthat encode a polypeptide, fragment of a polypeptide, peptide, or fusionpolypeptide that has at least one activity of an MVA pathwaypolypeptide. Exemplary MVA pathway polypeptides and nucleic acidsinclude naturally-occurring polypeptides and nucleic acids from any ofthe source organisms described herein as well as mutant polypeptides andnucleic acids derived from any of the source organisms described herein.

In particular, acetyl-CoA acetyltransferase polypeptides (AA-CoAthiolase or AACT) convert two molecules of acetyl-CoA intoacetoacetyl-CoA. Standard methods (such as those described herein) canbe used to determine whether a polypeptide has AA-CoA thiolasepolypeptide activity by measuring the ability of the polypeptide toconvert two molecules of acetyl-CoA into acetoacetyl-CoA in vitro, in acell extract, or in vivo.

3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase or HMGS)polypeptides convert acetoacetyl-CoA into3-hydroxy-3-methylglutaryl-CoA. Standard methods (such as thosedescribed herein) can be used to determine whether a polypeptide hasHMG-CoA synthase polypeptide activity by measuring the ability of thepolypeptide to convert acetoacetyl-CoA into3-hydroxy-3-methylglutaryl-CoA in vitro, in a cell extract, or in vivo.

3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase or HMGR)polypeptides convert 3-hydroxy-3-methylglutaryl-CoA into mevalonate.Standard methods (such as those described herein) can be used todetermine whether a polypeptide has HMG-CoA reductase polypeptideactivity by measuring the ability of the polypeptide to convert3-hydroxy-3-methylglutaryl-CoA into mevalonate in vitro, in a cellextract, or in vivo.

Mevalonate kinase (MVK) polypeptide phosphorylates mevalonate to formmevalonate-5-phosphate. Standard methods (such as those describedherein) can be used to determine whether a polypeptide has MVKpolypeptide activity by measuring the ability of the polypeptide toconvert mevalonate into mevalonate-5-phosphate in vitro, in a cellextract, or in vivo.

Phosphomevalonate kinase (PMK) polypeptides phosphorylatesmevalonate-5-phosphate to form mevalonate-5-diphosphate. Standardmethods (such as those described herein) can be used to determinewhether a polypeptide has PMK polypeptide activity by measuring theability of the polypeptide to convert mevalonate-5-phosphate intomevalonate-5-diphosphate in vitro, in a cell extract, or in vivo.

Diphosphomevalonte decarboxylase (MVD or DPMDC) polypeptides convertmevalonate-5-diphosphate into isopentenyl diphosphate (IPP). Standardmethods (such as those described herein) can be used to determinewhether a polypeptide has MVD polypeptide activity by measuring theability of the polypeptide to convert mevalonate-5-diphosphate into IPPin vitro, in a cell extract, or in vivo.

Phosphomevalonate decarboxylase (PMDC) polypeptides convertmevalonate-5-phosphate into isopentenyl phosphate (IP). Standard methods(such as those described herein) can be used to determine whether apolypeptide has PMDC polypeptide activity by measuring the ability ofthe polypeptide to convert mevalonate-5-phosphate into IP in vitro, in acell extract, or in vivo.

Isopentenyl phosphate kinase (IPK) polypeptides phosphorylate isopentylphosphate (IP) to form isopentenyl diphosphate (IPP). Standard methods(such as those described herein) can be used to determine whether apolypeptide has IPK polypeptide activity by measuring the ability of thepolypeptide to convert IP into IPP in vitro, in a cell extract, or invivo.

Exemplary IDI polypeptides and nucleic acids are described above.

Exemplary Methods for Isolating Nucleic Acids

Isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acids can beisolated using standard methods. Methods of obtaining desired nucleicacids from a source organism of interest (such as a bacterial genome)are common and well known in the art of molecular biology (see, forexample, WO 2004/033646 and references cited therein, which are eachhereby incorporated by reference in their entireties, particularly withrespect to the isolation of nucleic acids of interest). For example, ifthe sequence of the nucleic acid is known (such as any of the knownnucleic acids described herein), suitable genomic libraries may becreated by restriction endonuclease digestion and may be screened withprobes complementary to the desired nucleic acid sequence. Once thesequence is isolated, the DNA may be amplified using standard primerdirected amplification methods such as polymerase chain reaction (PCR)(U.S. Pat. No. 4,683,202, which is incorporated by reference in itsentirety, particularly with respect to PCR methods) to obtain amounts ofDNA suitable for transformation using appropriate vectors.

Alternatively, isoprene synthase, DXS, IDI, and/or MVA pathway nucleicacids (such as any isoprene synthase, DXS, IDI, and/or MVA pathwaynucleic acids with a known nucleic acid sequence) can be chemicallysynthesized using standard methods.

Additional isoprene synthase, DXS, IDI, or MVA pathway polypeptides andnucleic acids which may be suitable for use in the compositions andmethods described herein can be identified using standard methods. Forexample, cosmid libraries of the chromosomal DNA of organisms known toproduce isoprene naturally can be constructed in organisms such as E.coli, and then screened for isoprene production. In particular, cosmidlibraries may be created where large segments of genomic DNA (35-45 kb)are packaged into vectors and used to transform appropriate hosts.Cosmid vectors are unique in being able to accommodate large quantitiesof DNA. Generally cosmid vectors have at least one copy of the cos DNAsequence which is needed for packaging and subsequent circularization ofthe heterologous DNA. In addition to the cos sequence, these vectorsalso contain an origin of replication such as ColEI and drug resistancemarkers such as a nucleic acid resistant to ampicillin or neomycin.Methods of using cosmid vectors for the transformation of suitablebacterial hosts are well described in Sambrook et al., MolecularCloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor, 1989,which is hereby incorporated by reference in its entirety, particularlywith respect to transformation methods.

Typically to clone cosmids, heterologous DNA is isolated using theappropriate restriction endonucleases and ligated adjacent to the cosregion of the cosmid vector using the appropriate ligases. Cosmidvectors containing the linearized heterologous DNA are then reacted witha DNA packaging vehicle such as bacteriophage. During the packagingprocess, the cos sites are cleaved and the heterologous DNA is packagedinto the head portion of the bacterial viral particle. These particlesare then used to transfect suitable host cells such as E. coli. Onceinjected into the cell, the heterologous DNA circularizes under theinfluence of the cos sticky ends. In this manner, large segments ofheterologous DNA can be introduced and expressed in host cells.

Additional methods for obtaining isoprene synthase, DXS, IDI, and/or MVApathway nucleic acids include screening a metagenomic library by assay(such as the headspace assay described herein) or by PCR using primersdirected against nucleotides encoding for a length of conserved aminoacids (for example, at least 3 conserved amino acids). Conserved aminoacids can be identified by aligning amino acid sequences of knownisoprene synthase, DXS, IDI, and/or MVA pathway polypeptides. Conservedamino acids for isoprene synthase polypeptides can be identified basedon aligned sequences of known isoprene synthase polypeptides. Anorganism found to produce isoprene naturally can be subjected tostandard protein purification methods (which are well known in the art)and the resulting purified polypeptide can be sequenced using standardmethods. Other methods are found in the literature (see, for example,Julsing et al., Applied. Microbiol. Biotechnol. 75: 1377-84, 2007;Withers et al., Appl Environ Microbiol. 73(19):6277-83, 2007, which areeach hereby incorporated by reference in their entireties, particularlywith respect to identification of nucleic acids involved in thesynthesis of isoprene).

Additionally, standard sequence alignment and/or structure predictionprograms can be used to identify additional DXS, IDI, or MVA pathwaypolypeptides and nucleic acids based on the similarity of their primaryand/or predicted polypeptide secondary structure with that of known DXS,IDI, or MVA pathway polypeptides and nucleic acids. Standard databasessuch as the swissprot-trembl database (world-wide web at “expasy.org”,Swiss Institute of Bioinformatics Swiss-Prot group CMU-1 rue MichelServet CH-1211 Geneva 4, Switzerland) can also be used to identifyisoprene synthase, DXS, IDI, or MVA pathway polypeptides and nucleicacids. The secondary and/or tertiary structure of an isoprene synthase,DXS, IDI, or MVA pathway polypeptide can be predicted using the defaultsettings of standard structure prediction programs, such asPredictProtein (630 West, 168 Street, BB217, New York, N.Y. 10032, USA).Alternatively, the actual secondary and/or tertiary structure of anisoprene synthase, DXS, IDI, or MVA pathway polypeptide can bedetermined using standard methods. Additional isoprene synthase, DXS,IDI, or MVA pathway nucleic acids can also be identified byhybridization to probes generated from known isoprene synthase, DXS,IDI, or MVA pathway nucleic acids.

Exemplary Promoters and Vectors

Any of the isoprene synthase, DXS, IDI, or MVA pathway nucleic aciddescribed herein can be included in one or more vectors. Accordingly,the invention also features vectors with one more nucleic acids encodingany of the isoprene synthase, DXS, IDI, or MVA pathway polypeptides thatare described herein. As used herein, a “vector” means a construct thatis capable of delivering, and desirably expressing one or more nucleicacids of interest in a host cell. Examples of vectors include, but arenot limited to, plasmids, viral vectors, DNA or RNA expression vectors,cosmids, and phage vectors. In some embodiments, the vector contains anucleic acid under the control of an expression control sequence.

As used herein, an “expression control sequence” means a nucleic acidsequence that directs transcription of a nucleic acid of interest. Anexpression control sequence can be a promoter, such as a constitutive oran inducible promoter, or an enhancer. An “inducible promoter” is apromoter that is active under environmental or developmental regulation.The expression control sequence is operably linked to the nucleic acidsegment to be transcribed.

In some embodiments, the vector contains a selective marker. The term“selective marker” refers to a nucleic acid capable of expression in ahost cell that allows for ease of selection of those host cellscontaining an introduced nucleic acid or vector. Examples of selectablemarkers include, but are not limited to, antibiotic resistance nucleicacids (e.g., kanamycin, ampicillin, carbenicillin, gentamicin,hygromycin, phleomycin, bleomycin, neomycin, or chloramphenicol) and/ornucleic acids that confer a metabolic advantage, such as a nutritionaladvantage on the host cell. Exemplary nutritional selective markersinclude those markers known in the art as amdS, argB, and pyr4. Markersuseful in vector systems for transformation of Trichoderma are known inthe art (see, e.g., Finkelstein, Chapter 6 in Biotechnology ofFilamentous Fungi, Finkelstein et al., Eds. Butterworth-Heinemann,Boston, Mass., Chap. 6., 1992; and Kinghorn et al., Applied MolecularGenetics of Filamentous Fungi, Blackie Academic and Professional,Chapman and Hall, London, 1992, which are each hereby incorporated byreference in their entireties, particularly with respect to selectivemarkers). In some embodiments, the selective marker is the amdS nucleicacid, which encodes the enzyme acetamidase, allowing transformed cellsto grow on acetamide as a nitrogen source. The use of an A. nidulansamdS nucleic acid as a selective marker is described in Kelley et al.,EMBO J. 4:475-479, 1985 and Penttila et al., Gene 61:155-164, 1987(which are each hereby incorporated by reference in their entireties,particularly with respect to selective markers). In some embodiments, anisoprene synthase, DXS, IDI, or MVA pathway nucleic acid integrates intoa chromosome of the cells without a selective marker.

Suitable vectors are those which are compatible with the host cellemployed. Suitable vectors can be derived, for example, from abacterium, a virus (such as bacteriophage T7 or a M-13 derived phage), acosmid, a yeast, or a plant. Protocols for obtaining and using suchvectors are known to those in the art (see, for example, Sambrook etal., Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold SpringHarbor, 1989, which is hereby incorporated by reference in its entirety,particularly with respect to the use of vectors).

Promoters are well known in the art. Any promoter that functions in thehost cell can be used for expression of an isoprene synthase, DXS, IDI,or MVA pathway nucleic acid in the host cell. Initiation control regionsor promoters, which are useful to drive expression of isoprene synthase,DXS, IDI, or MVA pathway nucleic acids in various host cells arenumerous and familiar to those skilled in the art (see, for example, WO2004/033646 and references cited therein, which are each herebyincorporated by reference in their entireties, particularly with respectto vectors for the expression of nucleic acids of interest). Virtuallyany promoter capable of driving these nucleic acids is suitable for thepresent invention including, but not limited to, CYC1, HIS3, GAL1,GAL10, ADH1, PGK, PHO5, GAPDH, ADCI, TRP1, URA3, LEU2, ENO, and TPI(useful for expression in Saccharomyces); AOX1 (useful for expression inPichia); and lac, trp,

P_(L),

P_(R), T7, tac, and trc (useful for expression in E. coli).

In some embodiments, a glucose isomerase promoter is used (see, forexample, U.S. Pat. No. 7,132,527 and references cited therein, which areeach hereby incorporated by reference in their entireties, particularlywith respect promoters and plasmid systems for expressing polypeptidesof interest). Reported glucose isomerase promoter mutants can be used tovary the level of expression of the polypeptide encoded by a nucleicacid operably linked to the glucose isomerase promoter (U.S. Pat. No.7,132,527). In various embodiments, the glucose isomerase promoter iscontained in a low, medium, or high copy plasmid (U.S. Pat. No.7,132,527).

In various embodiments, an isoprene synthase, DXS, IDI, and/or MVApathway nucleic acid is contained in a low copy plasmid (e.g., a plasmidthat is maintained at about 1 to about 4 copies per cell), medium copyplasmid (e.g., a plasmid that is maintained at about 10 to about 15copies per cell), or high copy plasmid (e.g., a plasmid that ismaintained at about 50 or more copies per cell). In some embodiments,the heterologous or extra endogenous isoprene synthase, DXS, IDI, or MVApathway nucleic acid is operably linked to a T7 promoter. In someembodiments, the heterologous or extra endogenous isoprene synthase,DXS, IDI, or MVA pathway nucleic acid operably linked to a T7 promoteris contained in a medium or high copy plasmid. In some embodiments, theheterologous or extra endogenous isoprene synthase, DXS, IDI, or MVApathway nucleic acid is operably linked to a Trc promoter. In someembodiments, the heterologous or extra endogenous isoprene synthase,DXS, IDI, or MVA pathway nucleic acid operably linked to a Trc promoteris contained in a medium or high copy plasmid. In some embodiments, theheterologous or extra endogenous isoprene synthase, DXS, IDI, or MVApathway nucleic acid is operably linked to a Lac promoter. In someembodiments, the heterologous or extra endogenous isoprene synthase,DXS, IDI, or MVA pathway nucleic acid operably linked to a Lac promoteris contained in a low copy plasmid. In some embodiments, theheterologous or extra endogenous isoprene synthase, DXS, IDI, or MVApathway nucleic acid is operably linked to an endogenous promoter, suchas an endogenous Escherichia, Panteoa, Bacillus, Yarrowia, Streptomyces,or Trichoderma promoter or an endogenous alkaline serine protease,isoprene synthase, DXS, IDI, or MVA pathway promoter. In someembodiments, the heterologous or extra endogenous isoprene synthase,DXS, IDI, or MVA pathway nucleic acid operably linked to an endogenouspromoter is contained in a high copy plasmid. In some embodiments, thevector is a replicating plasmid that does not integrate into achromosome in the cells. In some embodiments, part or all of the vectorintegrates into a chromosome in the cells.

In some embodiments, the vector is any vector which when introduced intoa fungal host cell is integrated into the host cell genome and isreplicated. Reference is made to the Fungal Genetics Stock CenterCatalogue of Strains (FGSC, the world-wide web at “fgsc.net” and thereferences cited therein, which are each hereby incorporated byreference in their entireties, particularly with respect to vectors) fora list of vectors. Additional examples of suitable expression and/orintegration vectors are provided in Sambrook et al., Molecular Cloning:A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor, 1989, CurrentProtocols in Molecular Biology (F. M. Ausubel et al. (eds) 1987,Supplement 30, section 7.7.18); van den Hondel et al. in Bennett andLasure (Eds.) More Gene Manipulations in Fungi, Academic Press pp.396-428, 1991; and U.S. Pat. No. 5,874,276, which are each herebyincorporated by reference in their entireties, particularly with respectto vectors. Particularly useful vectors include pFB6, pBR322, PUC18,pUC100, and pENTR/D.

In some embodiments, an isoprene synthase, DXS, IDI, or MVA pathwaynucleic acid is operably linked to a suitable promoter that showstranscriptional activity in a fungal host cell. The promoter may bederived from one or more nucleic acids encoding a polypeptide that iseither endogenous or heterologous to the host cell. In some embodiments,the promoter is useful in a Trichoderma host. Suitable non-limitingexamples of promoters include cbh1, cbh2, egl1, egl2, pepA, hfb1, hfb2,xyn1, and amy. In some embodiments, the promoter is one that is nativeto the host cell. For example, in some embodiments when T. reesei is thehost, the promoter is a native T. reesei promoter. In some embodiments,the promoter is T. reesei cbh1, which is an inducible promoter and hasbeen deposited in GenBank under Accession No. D86235, which isincorporated by reference in its entirety, particularly with respect topromoters. In some embodiments, the promoter is one that is heterologousto the fungal host cell. Other examples of useful promoters includepromoters from the genes of A. awamori and A. niger glucoamylase (glaA)(Nunberg et al., Mol. Cell Biol. 4:2306-2315, 1984 and Boel et al., EMBOJ. 3:1581-1585, 1984, which are each hereby incorporated by reference intheir entireties, particularly with respect to promoters); Aspergillusniger alpha amylases, Aspergillus oryzae TAKA amylase, T. reesei xln1,and the T. reesei cellobiohydrolase 1 (EP 137280, which is incorporatedby reference in its entirety, particularly with respect to promoters).

In some embodiments, the expression vector also includes a terminationsequence. Termination control regions may also be derived from variousgenes native to the host cell. In some embodiments, the terminationsequence and the promoter sequence are derived from the same source. Inanother embodiment, the termination sequence is endogenous to the hostcell. A particularly suitable terminator sequence is cbh1 derived from aTrichoderma strain (such as T. reesei). Other useful fungal terminatorsinclude the terminator from an A. niger or A. awamori glucoamylasenucleic acid (Nunberg et al., Mol. Cell Biol. 4:2306-2315, 1984 and Boelet al., EMBO J. 3:1581-1585, 1984; which are each hereby incorporated byreference in their entireties, particularly with respect to fungalterminators). Optionally, a termination site may be included. Foreffective expression of the polypeptides, DNA encoding the polypeptideare linked operably through initiation codons to selected expressioncontrol regions such that expression results in the formation of theappropriate messenger RNA.

In some embodiments, the promoter, coding, region, and terminator alloriginate from the isoprene synthase, DXS, IDI, or MVA pathway nucleicacid to be expressed. In some embodiments, the coding region for anisoprene synthase, DXS, IDI, or MVA pathway nucleic acid is insertedinto a general-purpose expression vector such that it is under thetranscriptional control of the expression construct promoter andterminator sequences. In some embodiments, genes or part thereof areinserted downstream of the strong cbh1 promoter.

An isoprene synthase, DXS, IDI, or MVA pathway nucleic acid can beincorporated into a vector, such as an expression vector, using standardtechniques (Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor, 1982, which is hereby incorporated by reference inits entirety, particularly with respect to the screening of appropriateDNA sequences and the construction of vectors). Methods used to ligatethe DNA construct comprising a nucleic acid of interest (such as anisoprene synthase, DXS, IDI, or MVA pathway nucleic acid), a promoter, aterminator, and other sequences and to insert them into a suitablevector are well known in the art. For example, restriction enzymes canbe used to cleave the isoprene synthase, DXS, IDI, or MVA pathwaynucleic acid and the vector. Then, the compatible ends of the cleavedisoprene synthase, DXS, IDI, or MVA pathway nucleic acid and the cleavedvector can be ligated. Linking is generally accomplished by ligation atconvenient restriction sites. If such sites do not exist, the syntheticoligonucleotide linkers are used in accordance with conventionalpractice (see, Sambrook et al., Molecular Cloning: A Laboratory Manual,2^(nd) ed., Cold Spring Harbor, 1989, and Bennett and Lasure, More GeneManipulations in Fungi, Academic Press, San Diego, pp 70-76, 1991, whichare each hereby incorporated by reference in their entireties,particularly with respect to oligonucleotide linkers). Additionally,vectors can be constructed using known recombination techniques (e.g.,Invitrogen Life Technologies, Gateway Technology).

In some embodiments, it may be desirable to over-express isoprenesynthase, DXS, IDI, or MVA pathway nucleic acids at levels far higherthan currently found in naturally-occurring cells. This result may beaccomplished by the selective cloning of the nucleic acids encodingthose polypeptides into multicopy plasmids or placing those nucleicacids under a strong inducible or constitutive promoter. Methods forover-expressing desired polypeptides are common and well known in theart of molecular biology and examples may be found in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor,1989, which is hereby incorporated by reference in its entirety,particularly with respect to cloning techniques.

The following resources include descriptions of additional generalmethodology useful in accordance with the invention: Kreigler, GeneTransfer and Expression; A Laboratory Manual, 1990 and Ausubel et al.,Eds. Current Protocols in Molecular Biology, 1994, which are each herebyincorporated by reference in their entireties, particularly with respectto molecular biology and cloning techniques.

Exemplary Source Organisms

Isoprene synthase, DXS, IDI, or MVA pathway nucleic acids (and theirencoded polypeptides) can be obtained from any organism that naturallycontains isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acids.As noted above, isoprene is formed naturally by a variety of organisms,such as bacteria, yeast, plants, and animals. Organisms contain the MVApathway, DXP pathway, or both the MVA and DXP pathways for producingisoprene (FIG. 19). Thus, DXS nucleic acids can be obtained, e.g., fromany organism that contains the DXP pathway or contains both the MVA andDXP pathways. IDI and isoprene synthase nucleic acids can be obtained,e.g., from any organism that contains the MVA pathway, DXP pathway, orboth the MVA and DXP pathways. MVA pathway nucleic acids can beobtained, e.g., from any organism that contains the MVA pathway orcontains both the MVA and DXP pathways.

In some embodiments, the nucleic acid sequence of the isoprene synthase,DXS, IDI, or MVA pathway nucleic is identical to the sequence of anucleic acid that is produced by any of the following organisms innature. In some embodiments, the amino acid sequence of the isoprenesynthase, DXS, IDI, or MVA pathway polypeptide is identical to thesequence of a polypeptide that is produced by any of the followingorganisms in nature. In some embodiments, the isoprene synthase, DXS,IDI, or MVA pathway nucleic acid or polypeptide is a mutant nucleic acidor polypeptide derived from any of the organisms described herein. Asused herein, “derived from” refers to the source of the nucleic acid orpolypeptide into which one or more mutations is introduced. For example,a polypeptide that is “derived from a plant polypeptide” refers topolypeptide of interest that results from introducing one or moremutations into the sequence of a wild-type (i.e., a sequence occurringin nature) plant polypeptide.

In some embodiments, the source organism is a fungus, examples of whichare species of Aspergillus such as A. oryzae and A. niger, species ofSaccharomyces such as S. cerevisiae, species of Schizosaccharomyces suchas S. pombe, and species of Trichoderma such as T. reesei. In someembodiments, the source organism is a filamentous fungal cell. The term“filamentous fungi” refers to all filamentous forms of the subdivisionEumycotina (see, Alexopoulos, C. J. (1962), Introductory Mycology,Wiley, New York). These fungi are characterized by a vegetative myceliumwith a cell wall composed of chitin, cellulose, and other complexpolysaccharides. The filamentous fungi are morphologically,physiologically, and genetically distinct from yeasts. Vegetative growthby filamentous fungi is by hyphal elongation and carbon catabolism isobligatory aerobic. The filamentous fungal parent cell may be a cell ofa species of, but not limited to, Trichoderma, (e.g., Trichodermareesei, the asexual morph of Hypocrea jecorina, previously classified asT. longibrachiatum, Trichoderma viride, Trichoderma koningii,Trichoderma harzianum) (Sheir-Neirs et al., Appl. Microbiol. Biotechnol20: 46-53, 1984; ATCC No. 56765 and ATCC No. 26921); Penicillium sp.,Humicola sp. (e.g., H. insolens, H. lanuginose, or H. grisea);Chrysosporium sp. (e.g., C. lucknowense), Gliocladium sp., Aspergillussp. (e.g., A. oryzae, A. niger, A. sojae, A. japonicus, A. nidulans, orA. awamori) (Ward et al., Appl. Microbiol. Biotechnol. 39: 7380743, 1993and Goedegebuur et al., Genet 41: 89-98, 2002), Fusarium sp., (e.g., F.roseum, F. graminum F. cerealis, F. oxysporuim, or F. venenatum),Neurospora sp., (e.g., N. crassa), Hypocrea sp., Mucor sp., (e.g., M.miehei), Rhizopus sp. and Emericella sp. (see also, Innis et al., Sci.228: 21-26, 1985). The term “Trichoderma” or “Trichoderma sp.” or“Trichoderma spp.” refer to any fungal genus previously or currentlyclassified as Trichoderma.

In some embodiments, the fungus is A. nidulans, A. awamori, A. oryzae,A. aculeatus, A. niger, A. japonicus, T. reesei, T. viride, F.oxysporum, or F. solani. Aspergillus strains are disclosed in Ward etal., Appl. Microbiol. Biotechnol. 39:738-743, 1993 and Goedegebuur etal., Curr Gene 41:89-98, 2002, which are each hereby incorporated byreference in their entireties, particularly with respect to fungi. Inparticular embodiments, the fungus is a strain of Trichoderma, such as astrain of T. reesei. Strains of T. reesei are known and non-limitingexamples include ATCC No. 13631, ATCC No. 26921, ATCC No. 56764, ATCCNo. 56765, ATCC No. 56767, and NRRL 15709, which are each herebyincorporated by reference in their entireties, particularly with respectto strains of T. reesei. In some embodiments, the host strain is aderivative of RL-P37. RL-P37 is disclosed in Sheir-Neiss et al., Appl.Microbiol. Biotechnology 20:46-53, 1984, which is hereby incorporated byreference in its entirety, particularly with respect to strains of T.reesei.

In some embodiments, the source organism is a yeast, such asSaccharomyces sp., Schizosaccharomyces sp., Pichia sp., or Candida sp.

In some embodiments, the source organism is a bacterium, such as strainsof Bacillus such as B. lichenformis or B. subtilis, strains of Pantoeasuch as P. citrea, strains of Pseudomonas such as P. alcaligenes,strains of Streptomyces such as S. albus, S. lividans, or S.rubiginosus, or strains of Escherichia such as E. coli.

As used herein, “the genus Bacillus” includes all species within thegenus “Bacillus,” as known to those of skill in the art, including butnot limited to B. subtilis, B. licheniformis, B. lentus, B. brevis, B.stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii,B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, andB. thuringiensis. It is recognized that the genus Bacillus continues toundergo taxonomical reorganization. Thus, it is intended that the genusinclude species that have been reclassified, including but not limitedto such organisms as B. stearothermophilus, which is now named“Geobacillus stearothermophilus.” The production of resistant endosporesin the presence of oxygen is considered the defining feature of thegenus Bacillus, although this characteristic also applies to therecently named Alicyclobacillus, Amphibacillus, Aneurinibacillus,Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus,Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus,and Virgibacillus.

In some embodiments, the source organism is a gram-positive bacterium.Non-limiting examples include strains of Streptomyces (e.g., S. albus,S. lividans, S. coelicolor, or S. griseus) and Bacillus. In someembodiments, the source organism is a gram-negative bacterium, such asE. coli or Pseudomonas sp.

In some embodiments, the source organism is a plant, such as a plantfrom the family Fabaceae, such as the Faboideae subfamily. In someembodiments, the source organism is kudzu, poplar (such as Populusalba×tremula CAC35696 or Populus alba) (Sasaki et al., FEBS Letters579(11): 2514-2518, 2005), aspen (such as Populus tremuloides), orQuercus robur.

In some embodiments, the source organism is an algae, such as a greenalgae, red algae, glaucophytes, chlorarachniophytes, euglenids,chromista, or dinoflagellates.

In some embodiments, the source organism is a cyanobacteria, such ascyanobacteria classified into any of the following groups based onmorphology: Chroococcales, Pleurocapsales, Oscillatoriales, Nostocales,or Stigonematales.

Exemplary Host Cells

A variety of host cells can be used to express isoprene synthase, DXS,IDI, and/or MVA pathway polypeptides and to produce isoprene in themethods of the claimed invention. Exemplary host cells include cellsfrom any of the organisms listed in the prior section under the heading“Exemplary Source Organisms.” The host cell may be a cell that naturallyproduces isoprene or a cell that does not naturally produce isoprene. Insome embodiments, the host cell naturally produces isoprene using theDXP pathway, and an isoprene synthase, DXS, and/or IDI nucleic acid isadded to enhance production of isoprene using this pathway. In someembodiments, the host cell naturally produces isoprene using the MVApathway, and an isoprene synthase and/or one or more MVA pathway nucleicacids are added to enhance production of isoprene using this pathway. Insome embodiments, the host cell naturally produces isoprene using theDXP pathway and one or more MVA pathway nucleic acids are added toproduce isoprene using part or all of the MVA pathway as well as the DXPpathway. In some embodiments, the host cell naturally produces isopreneusing both the DXP and MVA pathways and one or more isoprene synthase,DXS, IDI, or MVA pathway nucleic acids are added to enhance productionof isoprene by one or both of these pathways.

Exemplary Transformation Methods

Isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acids or vectorscontaining them can be inserted into a host cell (e.g., a plant cell, afungal cell, a yeast cell, or a bacterial cell described herein) usingstandard techniques for expression of the encoded isoprene synthase,DXS, IDI, and/or MVA pathway polypeptide. Introduction of a DNAconstruct or vector into a host cell can be performed using techniquessuch as transformation, electroporation, nuclear microinjection,transduction, transfection (e.g., lipofection mediated or DEAE-Dextrinmediated transfection or transfection using a recombinant phage virus),incubation with calcium phosphate DNA precipitate, high velocitybombardment with DNA-coated microprojectiles, and protoplast fusion.General transformation techniques are known in the art (see, e.g.,Current Protocols in Molecular Biology (F. M. Ausubel et al. (eds)Chapter 9, 1987; Sambrook et al., Molecular Cloning: A LaboratoryManual, 2^(nd) ed., Cold Spring Harbor, 1989; and Campbell et al., Curr.Genet. 16:53-56, 1989, which are each hereby incorporated by referencein their entireties, particularly with respect to transformationmethods). The expression of heterologous polypeptide in Trichoderma isdescribed in U.S. Pat. No. 6,022,725; U.S. Pat. No. 6,268,328; U.S. Pat.No. 7,262,041; WO 2005/001036; Harkki et al.; Enzyme Microb. Technol.13:227-233, 1991; Harkki et al., Bio Technol. 7:596-603, 1989; EP244,234; EP 215,594; and Nevalainen et al., “The Molecular Biology ofTrichoderma and its Application to the Expression of Both Homologous andHeterologous Genes, ” in Molecular Industrial Mycology, Eds. Leong andBerka, Marcel Dekker Inc., NY pp. 129-148, 1992, which are each herebyincorporated by reference in their entireties, particularly with respectto transformation and expression methods). Reference is also made to Caoet al., (Sci. 9:991-1001, 2000; EP 238023; and Yelton et al.,Proceedings. Natl. Acad. Sci. USA 81:1470-1474, 1984 (which are eachhereby incorporated by reference in their entireties, particularly withrespect to transformation methods) for transformation of Aspergillusstrains. The introduced nucleic acids may be integrated into chromosomalDNA or maintained as extrachromosomal replicating sequences.

Any method known in the art may be used to select transformants. In onenon-limiting example, stable transformants including an amdS marker aredistinguished from unstable transformants by their faster growth rateand the formation of circular colonies with a smooth, rather than raggedoutline on solid culture medium containing acetamide. Additionally, insome cases a further test of stability is conducted by growing thetransformants on a solid non-selective medium (e.g., a medium that lacksacetamide), harvesting spores from this culture medium, and determiningthe percentage of these spores which subsequently germinate and grow onselective medium containing acetamide.

In some embodiments, fungal cells are transformed by a process involvingprotoplast formation and transformation of the protoplasts followed byregeneration of the cell wall in a known manner. In one specificembodiment, the preparation of Trichoderma sp. for transformationinvolves the preparation of protoplasts from fungal mycelia (see,Campbell et al., Curr. Genet. 16:53-56, 1989, which is incorporated byreference in its entirety, particularly with respect to transformationmethods). In some embodiments, the mycelia are obtained from germinatedvegetative spores. The mycelia are treated with an enzyme that digeststhe cell wall resulting in protoplasts. The protoplasts are thenprotected by the presence of an osmotic stabilizer in the suspendingmedium. These stabilizers include sorbitol, mannitol, potassiumchloride, magnesium sulfate, and the like. Usually the concentration ofthese stabilizers varies between 0.8 M and 1.2 M. It is desirable to useabout a 1.2 M solution of sorbitol in the suspension medium.

Uptake of DNA into the host Trichoderma sp. strain is dependent upon thecalcium ion concentration. Generally, between about 10 mM CaCl₂ and 50mM CaCl₂ is used in an uptake solution. In addition to the calcium ionin the uptake solution, other compounds generally included are abuffering system such as TE buffer (10 Mm Tris, pH 7.4; 1 mM EDTA) or 10mM MOPS, pH 6.0 buffer (morpholinepropanesulfonic acid) and polyethyleneglycol (PEG). While not intending to be bound to any particular theory,it is believed that the polyethylene glycol acts to fuse the cellmembranes, thus permitting the contents of the medium to be deliveredinto the cytoplasm of the Trichoderma sp. strain and the plasmid DNA tobe transferred to the nucleus. This fusion frequently leaves multiplecopies of the plasmid DNA integrated into the host chromosome.

Usually a suspension containing the Trichoderma sp. protoplasts or cellsthat have been subjected to a permeability treatment at a density of 10⁵to 10⁷/mL (such as 2×10⁶/mL) are used in the transformation. A volume of100 μL of these protoplasts or cells in an appropriate solution (e.g.,1.2 M sorbitol and 50 mM CaCl₂) are mixed with the desired DNA.Generally, a high concentration of PEG is added to the uptake solution.From 0.1 to 1 volume of 25% PEG 4000 can be added to the protoplastsuspension. In some embodiments, about 0.25 volumes are added to theprotoplast suspension. Additives such as dimethyl sulfoxide, heparin,spermidine, potassium chloride, and the like may also be added to theuptake solution and aid in transformation. Similar procedures areavailable for other fungal host cells (see, e.g., U.S. Pat. Nos.6,022,725 and 6,268,328, which are each hereby incorporated by referencein their entireties, particularly with respect to transformationmethods).

Generally, the mixture is then cultured at approximately 0° C. for aperiod of between 10 to 30 minutes. Additional PEG is then added to themixture to further enhance the uptake of the desired nucleic acidsequence. The 25% PEG 4000 is generally added in volumes of 5 to 15times the volume of the transformation mixture; however, greater andlesser volumes may be suitable. The 25% PEG 4000 is desirably about 10times the volume of the transformation mixture. After the PEG is added,the transformation mixture is then cultured either at room temperatureor on ice before the addition of a sorbitol and CaCl₂ solution. Theprotoplast suspension is then further added to molten aliquots of agrowth medium. When the growth medium includes a growth selection (e.g.,acetamide or an antibiotic) it permits the growth of transformants only.

The transformation of bacterial cells may be performed according toconventional methods, e.g., as described in Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor, 1982, which is herebyincorporated by reference in its entirety, particularly with respect totransformation methods.

Exemplary Cell Culture Media

The invention also includes a cell or a population of cells in culturethat produce isoprene. By “cells in culture” is meant two or more cellsin a solution (e.g., a cell medium) that allows the cells to undergo oneor more cell divisions. “Cells in culture” do not include plant cellsthat are part of a living, multicellular plant containing cells thathave differentiated into plant tissues. In various embodiments, the cellculture includes at least or about 10, 20, 50, 100, 200, 500, 1,000,5,000, 10,000 or more cells.

Any carbon source can be used to cultivate the host cells. The term“carbon source” refers to one or more carbon-containing compoundscapable of being metabolized by a host cell or organism. For example,the cell medium used to cultivate the host cells may include any carbonsource suitable for maintaining the viability or growing the host cells.

In some embodiments, the carbon source is a carbohydrate (such asmonosaccharide, disaccharide, oligosaccharide, or polysaccharids),invert sugar (e.g., enzymatically treated sucrose syrup), glycerol,glycerine (e.g., a glycerine byproduct of a biodiesel or soap-makingprocess), dihydroxyacetone, one-carbon source, oil (e.g., a plant orvegetable oil such as corn, palm, or soybean oil), acetate, animal fat,animal oil, fatty acid (e.g., a saturated fatty acid, unsaturated fattyacid, or polyunsaturated fatty acid), lipid, phospholipid, glycerolipid,monoglyceride, diglyceride, triglyceride, polypeptide (e.g., a microbialor plant protein or peptide), renewable carbon source (e.g., a biomasscarbon source such as a hydrolyzed biomass carbon source), yeastextract, component from a yeast extract, polymer, acid, alcohol,aldehyde, ketone, amino acid, succinate, lactate, acetate, ethanol, orany combination of two or more of the foregoing. In some embodiments,the carbon source is a product of photosynthesis, including, but notlimited to, glucose. In some embodiment, the carbohydrate is xylose orglucose.

Exemplary monosaccharides include glucose and fructose; exemplaryoligosaccharides include lactose and sucrose, and exemplarypolysaccharides include starch and cellulose. Exemplary carbohydratesinclude C6 sugars (e.g., fructose, mannose, galactose, or glucose) andC5 sugars (e.g., xylose or arabinose). In some embodiments, the cellmedium includes a carbohydrate as well as a carbon source other than acarbohydrate (e.g., glycerol, glycerine, dihydroxyacetone, one-carbonsource, oil, animal fat, animal oil, fatty acid, lipid, phospholipid,glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbonsource, or a component from a yeast extract). In some embodiments, thecell medium includes a carbohydrate as well as a polypeptide (e.g., amicrobial or plant protein or peptide). In some embodiments, themicrobial polypeptide is a polypeptide from yeast or bacteria. In someembodiments, the plant polypeptide is a polypeptide from soy, corn,canola, jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed,cottonseed, palm kernel, olive, safflower, sesame, or linseed.

In some embodiments, the concentration of the carbohydrate is at leastor about 5 grams per liter of broth (g/L, wherein the volume of brothincludes both the volume of the cell medium and the volume of thecells), such as at least or about 10, 15, 20, 30, 40, 50, 60, 80, 100,150, 200, 300, 400, or more g/L. In some embodiments, the concentrationof the carbohydrate is between about 50 and about 400 g/L, such asbetween about 100 and about 360 g/L, between about 120 and about 360g/L, or between about 200 and about 300 g/L. In some embodiments, thisconcentration of carbohydrate includes the total amount of carbohydratethat is added before and/or during the culturing of the host cells.

Exemplary lipids are any substance containing one or more fatty acidsthat are C4 and above fatty acids that are saturated, unsaturated, orbranched.

Exemplary oils are lipids that are liquid at room temperature. In someembodiments, the lipid contains one or more C4 or above fatty acids(e.g., contains one or more saturated, unsaturated, or branched fattyacid with four or more carbons). In some embodiments, the oil isobtained from soy, corn, canola, jatropha, palm, peanut, sunflower,coconut, mustard, rapeseed, cottonseed, palm kernel, olive, safflower,sesame, linseed, oleagineous microbial cells, Chinese tallow, or anycombination of two or more of the foregoing.

Exemplary fatty acids include compounds of the formula RCOOH, where “R”is a hydrocarbon. Exemplary unsaturated fatty acids include compoundswhere “R” includes at least one carbon-carbon double bond. Exemplaryunsaturated fatty acids include, but are not limited to, oleic acid,vaccenic acid, linoleic acid, palmitelaidic acid, and arachidonic acid.Exemplary polyunsaturated fatty acids include compounds where “R”includes a plurality of carbon-carbon double bonds. Exemplary saturatedfatty acids include compounds where “R” is a saturated aliphatic group.In some embodiments, the carbon source includes one or more C₁₂-C₂₂fatty acids, such as a C₁₂ saturated fatty acid, a C₁₄ saturated fattyacid, a C₁₆ saturated fatty acid, a C₁₈ saturated fatty acid, a C₂₀saturated fatty acid, or a C₂₂ saturated fatty acid. In an exemplaryembodiment, the fatty acid is palmitic acid. In some embodiments, thecarbon source is a salt of a fatty acid (e.g., an unsaturated fattyacid), a derivative of a fatty acid (e.g., an unsaturated fatty acid),or a salt of a derivative of fatty acid (e.g., an unsaturated fattyacid). Suitable salts include, but are not limited to, lithium salts,potassium salts, sodium salts, and the like. Di- and triglycerols arefatty acid esters of glycerol.

In some embodiments, the concentration of the lipid, oil, fat, fattyacid, monoglyceride, diglyceride, or triglyceride is at least or about 1gram per liter of broth (g/L, wherein the volume of broth includes boththe volume of the cell medium and the volume of the cells), such as atleast or about 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, 150, 200, 300,400, or more g/L. In some embodiments, the concentration of the lipid,oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride isbetween about 10 and about 400 g/L, such as between about 25 and about300 g/L, between about 60 and about 180 g/L, or between about 75 andabout 150 g/L. In some embodiments, the concentration includes the totalamount of the lipid, oil, fat, fatty acid, monoglyceride, diglyceride,or triglyceride that is added before and/or during the culturing of thehost cells. In some embodiments, the carbon source includes both (i) alipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglycerideand (ii) a carbohydrate, such as glucose. In some embodiments, the ratioof the lipid, oil, fat, fatty acid, monoglyceride, diglyceride, ortriglyceride to the carbohydrate is about 1:1 on a carbon basis (i.e.,one carbon in the lipid, oil, fat, fatty acid, monoglyceride,diglyceride, or triglyceride per carbohydrate carbon). In particularembodiments, the amount of the lipid, oil, fat, fatty acid,monoglyceride, diglyceride, or triglyceride is between about 60 and 180g/L, and the amount of the carbohydrate is between about 120 and 360g/L.

Exemplary microbial polypeptide carbon sources include one or morepolypeptides from yeast or bacteria. Exemplary plant polypeptide carbonsources include one or more polypeptides from soy, corn, canola,jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed,cottonseed, palm kernel, olive, safflower, sesame, or linseed.

Exemplary renewable carbon sources include cheese whey permeate,cornsteep liquor, sugar beet molasses, barley malt, and components fromany of the foregoing. Exemplary renewable carbon sources also includeacetate, glucose, hexose, pentose and xylose present in biomass, such ascorn, switchgrass, sugar cane, cell waste of fermentation processes, andprotein by-product from the milling of soy, corn, or wheat. In someembodiments, the biomass carbon source is a lignocellulosic,hemicellulosic, or cellulosic material such as, but are not limited to,a grass, wheat, wheat straw, bagasse, sugar cane bagasse, soft woodpulp, corn, corn cob or husk, corn kernel, fiber from corn kernels, cornstover, switch grass, rice hull product, or a by-product from wet or drymilling of grains (e.g., corn, sorghum, rye, triticate, barley, wheat,and/or distillers grains). Exemplary cellulosic materials include wood,paper and pulp waste, herbaceous plants, and fruit pulp. In someembodiments, the carbon source includes any plant part, such as stems,grains, roots, or tubers. In some embodiments, all or part of any of thefollowing plants are used as a carbon source: corn, wheat, rye, sorghum,triticate, rice, millet, barley, cassava, legumes, such as beans andpeas, potatoes, sweet potatoes, bananas, sugarcane, and/or tapioca. Insome embodiments, the carbon source is a biomass hydrolysate, such as abiomass hydrolysate that includes both xylose and glucose or thatincludes both sucrose and glucose.

In some embodiments, the renewable carbon source (such as biomass) ispretreated before it is added to the cell culture medium. In someembodiments, the pretreatment includes enzymatic pretreatment, chemicalpretreatment, or a combination of both enzymatic and chemicalpretreatment (see, for example, Farzaneh et al., Bioresource Technology96 (18): 2014-2018, 2005; U.S. Pat. No. 6,176,176; U.S. Pat. No.6,106,888; which are each hereby incorporated by reference in theirentireties, particularly with respect to the pretreatment of renewablecarbon sources). In some embodiments, the renewable carbon source ispartially or completely hydrolyzed before it is added to the cellculture medium.

In some embodiments, the renewable carbon source (such as corn stover)undergoes ammonia fiber expansion (AFEX) pretreatment before it is addedto the cell culture medium (see, for example, Farzaneh et al.,Bioresource Technology 96 (18): 2014-2018, 2005). During AFEXpretreatment, a renewable carbon source is treated with liquid anhydrousammonia at moderate temperatures (such as about 60 to about 100° C.) andhigh pressure (such as about 250 to about 300 psi) for about 5 minutes.Then, the pressure is rapidly released. In this process, the combinedchemical and physical effects of lignin solubilization, hemicellulosehydrolysis, cellulose decrystallization, and increased surface areaenables near complete enzymatic conversion of cellulose andhemicellulose to fermentable sugars. AFEX pretreatment has the advantagethat nearly all of the ammonia can be recovered and reused, while theremaining serves as nitrogen source for microbes in downstreamprocesses. Also, a wash stream is not required for AFEX pretreatment.Thus, dry matter recovery following the AFEX treatment is essentially100%. AFEX is basically a dry to dry process. The treated renewablecarbon source is stable for long periods and can be fed at very highsolid loadings in enzymatic hydrolysis or fermentation processes.Cellulose and hemicellulose are well preserved in the AFEX process, withlittle or no degradation. There is no need for neutralization prior tothe enzymatic hydrolysis of a renewable carbon source that has undergoneAFEX pretreatment. Enzymatic hydrolysis of AFEX-treated carbon sourcesproduces clean sugar streams for subsequent fermentation use.

In some embodiments, the concentration of the carbon source (e.g., arenewable carbon source) is equivalent to at least or about 0.1, 0.5, 1,1.5, 2, 3, 4, 5, 10, 15, 20, 30, 40, or 50% glucose (w/v). Theequivalent amount of glucose can be determined by using standard HPLCmethods with glucose as a reference to measure the amount of glucosegenerated from the carbon source. In some embodiments, the concentrationof the carbon source (e.g., a renewable carbon source) is equivalent tobetween about 0.1 and about 20% glucose, such as between about 0.1 andabout 10% glucose, between about 0.5 and about 10% glucose, betweenabout 1 and about 10% glucose, between about 1 and about 5% glucose, orbetween about 1 and about 2% glucose.

In some embodiments, the carbon source includes yeast extract or one ormore components of yeast extract. In some embodiments, the concentrationof yeast extract is at least 1 gram of yeast extract per liter of broth(g/L, wherein the volume of broth includes both the volume of the cellmedium and the volume of the cells), such at least or about 5, 10, 15,20, 30, 40, 50, 60, 80, 100, 150, 200, 300, or more g/L. In someembodiments, the concentration of yeast extract is between about 1 andabout 300 g/L, such as between about 1 and about 200 g/L, between about5 and about 200 g/L, between about 5 and about 100 g/L, or between about5 and about 60 g/L. In some embodiments, the concentration includes thetotal amount of yeast extract that is added before and/or during theculturing of the host cells. In some embodiments, the carbon sourceincludes both yeast extract (or one or more components thereof) andanother carbon source, such as glucose. In some embodiments, the ratioof yeast extract to the other carbon source is about 1:5, about 1:10, orabout 1:20 (w/w).

Additionally the carbon source may also be one-carbon substrates such ascarbon dioxide, or methanol. Glycerol production from single carbonsources (e.g., methanol, formaldehyde, or formate) has been reported inmethylotrophic yeasts (Yamada et al., Agric. Biol. Chem., 53(2) 541-543,1989, which is hereby incorporated by reference in its entirety,particularly with respect to carbon sources) and in bacteria (Hunter et.al., Biochemistry, 24, 4148-4155, 1985, which is hereby incorporated byreference in its entirety, particularly with respect to carbon sources).These organisms can assimilate single carbon compounds, ranging inoxidation state from methane to formate, and produce glycerol. Thepathway of carbon assimilation can be through ribulose monophosphate,through serine, or through xylulose-momophosphate (Gottschalk, BacterialMetabolism, Second Edition, Springer-Verlag: New York, 1986, which ishereby incorporated by reference in its entirety, particularly withrespect to carbon sources). The ribulose monophosphate pathway involvesthe condensation of formate with ribulose-5-phosphate to form a sixcarbon sugar that becomes fructose and eventually the three carbonproduct glyceraldehyde-3-phosphate. Likewise, the serine pathwayassimilates the one-carbon compound into the glycolytic pathway viamethylenetetrahydrofolate.

In addition to one and two carbon substrates, methylotrophic organismsare also known to utilize a number of other carbon containing compoundssuch as methylamine, glucosamine and a variety of amino acids formetabolic activity. For example, methylotrophic yeast are known toutilize the carbon from methylamine to form trehalose or glycerol(Bellion et al., Microb. Growth Cl Compd., [Int. Symp.], 7^(th) ed.,415-32. Editors: Murrell et al., Publisher: Intercept, Andover, UK,1993, which is hereby incorporated by reference in its entirety,particularly with respect to carbon sources). Similarly, various speciesof Candida metabolize alanine or oleic acid (Sulter et al., Arch.Microbiol. 153(5), 485-9, 1990, which is hereby incorporated byreference in its entirety, particularly with respect to carbon sources).

In some embodiments, cells are cultured in a standard medium containingphysiological salts and nutrients (see, e.g., Pourquie, J. et al.,Biochemistry and Genetics of Cellulose Degradation, eds. Aubert et al.,Academic Press, pp. 71-86, 1988 and Ilmen et al., Appl. Environ.Microbiol. 63:1298-1306, 1997, which are each hereby incorporated byreference in their entireties, particularly with respect to cell media).Exemplary growth media are common commercially prepared media such asLuria Bertani (LB) broth, Sabouraud Dextrose (SD) broth, or Yeast medium(YM) broth. Other defined or synthetic growth media may also be used,and the appropriate medium for growth of particular host cells are knownby someone skilled in the art of microbiology or fermentation science.

In addition to an appropriate carbon source, the cell medium desirablycontains suitable minerals, salts, cofactors, buffers, and othercomponents known to those skilled in the art suitable for the growth ofthe cultures or the enhancement of isoprene production (see, forexample, WO 2004/033646 and references cited therein and WO 96/35796 andreferences cited therein, which are each hereby incorporated byreference in their entireties, particularly with respect cell medias andcell culture conditions). In some embodiments where an isoprenesynthase, DXS, IDI, and/or MVA pathway nucleic acid is under the controlof an inducible promoter, the inducing agent (e.g., a sugar, metal saltor antimicrobial), is desirably added to the medium at a concentrationeffective to induce expression of an isoprene synthase, DXS, IDI, and/orMVA pathway polypeptide. In some embodiments, cell medium has anantibiotic (such as kanamycin) that corresponds to the antibioticresistance nucleic acid (such as a kanamycin resistance nucleic acid) ona vector that has one or more DXS, IDI, or MVA pathway nucleic acids.

Exemplary Cell Culture Conditions

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Exemplary techniques maybe found in Manual of Methods for General Bacteriology Gerhardt et al.,eds), American Society for Microbiology, Washington, D.C. (1994) orBrock in Biotechnology: A Textbook of Industrial Microbiology, SecondEdition (1989) Sinauer Associates, Inc., Sunderland, Mass., which areeach hereby incorporated by reference in their entireties, particularlywith respect to cell culture techniques. In some embodiments, the cellsare cultured in a culture medium under conditions permitting theexpression of one or more isoprene synthase, DXS, IDI, or MVA pathwaypolypeptides encoded by a nucleic acid inserted into the host cells.

Standard cell culture conditions can be used to culture the cells (see,for example, WO 2004/033646 and references cited therein, which are eachhereby incorporated by reference in their entireties, particularly withrespect to cell culture and fermentation conditions). Cells are grownand maintained at an appropriate temperature, gas mixture, and pH (suchas at about 20 to about 37° C., at about 6% to about 84% CO₂, and at apH between about 5 to about 9). In some embodiments, cells are grown at35° C. in an appropriate cell medium. In some embodiments, e.g.,cultures are cultured at approximately 28° C. in appropriate medium inshake cultures or fermentors until desired amount of isoprene productionis achieved. In some embodiments, the pH ranges for fermentation arebetween about pH 5.0 to about pH 9.0 (such as about pH 6.0 to about pH8.0 or about 6.5 to about 7.0). Reactions may be performed underaerobic, anoxic, or anaerobic conditions based on the requirements ofthe host cells. Exemplary culture conditions for a given filamentousfungus are known in the art and may be found in the scientificliterature and/or from the source of the fungi such as the American TypeCulture Collection and Fungal Genetics Stock Center.

In various embodiments, the cells are grown using any known mode offermentation, such as batch, fed-batch, or continuous processes. In someembodiments, a batch method of fermentation is used. Classical batchfermentation is a closed system where the composition of the media isset at the beginning of the fermentation and is not subject toartificial alterations during the fermentation. Thus, at the beginningof the fermentation the cell medium is inoculated with the desired hostcells and fermentation is permitted to occur adding nothing to thesystem. Typically, however, “batch” fermentation is batch with respectto the addition of carbon source and attempts are often made atcontrolling factors such as pH and oxygen concentration. In batchsystems, the metabolite and biomass compositions of the system changeconstantly until the time the fermentation is stopped. Within batchcultures, cells moderate through a static lag phase to a high growth logphase and finally to a stationary phase where growth rate is diminishedor halted. In some embodiments, cells in log phase are responsible forthe bulk of the isoprene production. In some embodiments, cells instationary phase produce isoprene.

In some embodiments, a variation on the standard batch system is used,such as the Fed-Batch system. Fed-Batch fermentation processes comprisea typical batch system with the exception that the carbon source isadded in increments as the fermentation progresses. Fed-Batch systemsare useful when catabolite repression is apt to inhibit the metabolismof the cells and where it is desirable to have limited amounts of carbonsource in the cell medium. Fed-batch fermentations may be performed withthe carbon source (e.g., glucose) in a limited or excess amount.Measurement of the actual carbon source concentration in Fed-Batchsystems is difficult and is therefore estimated on the basis of thechanges of measurable factors such as pH, dissolved oxygen, and thepartial pressure of waste gases such as CO₂. Batch and Fed-Batchfermentations are common and well known in the art and examples may befound in Brock, Biotechnology: A Textbook of Industrial Microbiology,Second Edition (1989) Sinauer Associates, Inc., which is herebyincorporated by reference in its entirety, particularly with respect tocell culture and fermentation conditions.

In some embodiments, continuous fermentation methods are used.Continuous fermentation is an open system where a defined fermentationmedium is added continuously to a bioreactor and an equal amount ofconditioned medium is removed simultaneously for processing. Continuousfermentation generally maintains the cultures at a constant high densitywhere cells are primarily in log phase growth.

Continuous fermentation allows for the modulation of one factor or anynumber of factors that affect cell growth or isoprene production. Forexample, one method maintains a limiting nutrient such as the carbonsource or nitrogen level at a fixed rate and allows all other parametersto moderate. In other systems, a number of factors affecting growth canbe altered continuously while the cell concentration (e.g., theconcentration measured by media turbidity) is kept constant. Continuoussystems strive to maintain steady state growth conditions. Thus, thecell loss due to media being drawn off is balanced against the cellgrowth rate in the fermentation. Methods of modulating nutrients andgrowth factors for continuous fermentation processes as well astechniques for maximizing the rate of product formation are well knownin the art of industrial microbiology and a variety of methods aredetailed by Brock, Biotechnology: A Textbook of Industrial Microbiology,Second Edition (1989) Sinauer Associates, Inc., which is herebyincorporated by reference in its entirety, particularly with respect tocell culture and fermentation conditions.

In some embodiments, cells are immobilized on a substrate as whole cellcatalysts and subjected to fermentation conditions for isopreneproduction.

In some embodiments, bottles of liquid culture are placed in shakers inorder to introduce oxygen to the liquid and maintain the uniformity ofthe culture. In some embodiments, an incubator is used to control thetemperature, humidity, shake speed, and/or other conditions in which aculture is grown. The simplest incubators are insulated boxes with anadjustable heater, typically going up to ˜65° C. More elaborateincubators can also include the ability to lower the temperature (viarefrigeration), or the ability to control humidity or CO₂ levels. Mostincubators include a timer; some can also be programmed to cycle throughdifferent temperatures, humidity levels, etc. Incubators can vary insize from tabletop to units the size of small rooms.

If desired, a portion or all of the cell medium can be changed toreplenish nutrients and/or avoid the build up of potentially harmfulmetabolic byproducts and dead cells. In the case of suspension cultures,cells can be separated from the media by centrifuging or filtering thesuspension culture and then resuspending the cells in fresh media. Inthe case of adherent cultures, the media can be removed directly byaspiration and replaced. In some embodiments, the cell medium allows atleast a portion of the cells to divide for at least or about 5, 10, 20,40, 50, 60, 65, or more cell divisions in a continuous culture (such asa continuous culture without dilution).

In some embodiments, a constitutive or leaky promoter (such as a Trcpromoter) is used and a compound (such as IPTG) is not added to induceexpression of the isoprene synthase, DXS, IDI, or MVA pathway nucleicacid(s) operably linked to the promoter. In some embodiments, a compound(such as IPTG) is added to induce expression of the isoprene synthase,DXS, IDI, or MVA pathway nucleic acid(s) operably linked to thepromoter.

Exemplary Production of Isoprene

In some embodiments, the cells are cultured in a culture medium underconditions permitting the production of isoprene by the cells. By “peakabsolute productivity” is meant the maximum absolute amount of isoprenein the off-gas during the culturing of cells for a particular period oftime (e.g., the culturing of cells during a particular fermentationrun). By “peak absolute productivity time point” is meant the time pointduring a fermentation run when the absolute amount of isoprene in theoff-gas is at a maximum during the culturing of cells for a particularperiod of time (e.g., the culturing of cells during a particularfermentation run). In some embodiments, the isoprene amount is measuredat the peak absolute productivity time point. In some embodiments, thepeak absolute productivity for the cells is about any of the isopreneamounts disclosed herein.

By “peak specific productivity” is meant the maximum amount of isopreneproduced per cell during the culturing of cells for a particular periodof time (e.g., the culturing of cells during a particular fermentationrun). By “peak specific productivity time point” is meant the time pointduring the culturing of cells for a particular period of time (e.g., theculturing of cells during a particular fermentation run) when the amountof isoprene produced per cell is at a maximum. The peak specificproductivity is determined by dividing the total productivity by theamount of cells, as determined by optical density at 600 nm (OD₆₀₀). Insome embodiments, the isoprene amount is measured at the peak specificproductivity time point. In some embodiments, the peak specificproductivity for the cells is about any of the isoprene amounts per celldisclosed herein.

By “peak volumetric productivity” is meant the maximum amount ofisoprene produced per volume of broth (including the volume of the cellsand the cell medium) during the culturing of cells for a particularperiod of time (e.g., the culturing of cells during a particularfermentation run). By “peak specific volumetric productivity time point”is meant the time point during the culturing of cells for a particularperiod of time (e.g., the culturing of cells during a particularfermentation run) when the amount of isoprene produced per volume ofbroth is at a maximum. The peak specific volumetric productivity isdetermined by dividing the total productivity by the volume of broth andamount of time. In some embodiments, the isoprene amount is measured atthe peak specific volumetric productivity time point. In someembodiments, the peak specific volumetric productivity for the cells isabout any of the isoprene amounts per volume per time disclosed herein.

By “peak concentration” is meant the maximum amount of isoprene producedduring the culturing of cells for a particular period of time (e.g., theculturing of cells during a particular fermentation run). By “peakconcentration time point” is meant the time point during the culturingof cells for a particular period of time (e.g., the culturing of cellsduring a particular fermentation run) when the amount of isopreneproduced per cell is at a maximum. In some embodiments, the isopreneamount is measured at the peak concentration time point. In someembodiments, the peak concentration for the cells is about any of theisoprene amounts disclosed herein.

By “average volumetric productivity” is meant the average amount ofisoprene produced per volume of broth (including the volume of the cellsand the cell medium) during the culturing of cells for a particularperiod of time (e.g., the culturing of cells during a particularfermentation run). The average volumetric productivity is determined bydividing the total productivity by the volume of broth and amount oftime. In some embodiments, the average specific volumetric productivityfor the cells is about any of the isoprene amounts per volume per timedisclosed herein.

By “cumulative total productivity” is meant the cumulative, total amountof isoprene produced during the culturing of cells for a particularperiod of time (e.g., the culturing of cells during a particularfermentation run). In some embodiments, the cumulative, total amount ofisoprene is measured. In some embodiments, the cumulative totalproductivity for the cells is about any of the isoprene amountsdisclosed herein.

In some embodiments, the cells in culture produce isoprene at greaterthan or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600,700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000,5,000, 10,000, 12,500, 20,000, 30,000, 40,000, 50,000, 75,000, 100,000,125,000, 150,000, 188,000, or more nmole of isoprene/gram of cells forthe wet weight of the cells/hour (nmole/g_(wcm)/hr). In someembodiments, the amount of isoprene is between about 2 to about 200,000nmole/g_(wcm)/hr, such as between about 2 to about 100 nmole/g_(wcm)/hr,about 100 to about 500 nmole/g_(wcm)/hr, about 150 to about 500nmole/g_(wcm)/hr, about 500 to about 1,000 nmole/g_(wcm)/hr, about 1,000to about 2,000 nmole/g_(wcm)/hr, about 2,000 to about 5,000nmole/g_(wcm)/hr, about 5,000 to about 10,000 nmole/g_(wcm)/hr, about10,000 to about 50,000 nmole/g_(wcm)/hr, about 50,000 to about 100,000nmole/g_(wcm)/hr, about 100,000 to about 150,000 nmole/g_(wcm)/hr, orabout 150,000 to about 200,000 nmole/g_(wcm)/hr. In some embodiments,the amount of isoprene is between about 20 to about 200,000nmole/g_(wcm)/hr, about 100 to about 5,000 nmole/g_(wcm)/hr, about 200to about 2,000 nmole/g_(wcm)/hr, about 200 to about 1,000nmole/g_(wcm)/hr, about 300 to about 1,000 nmole/g_(wcm)/hr, about 400to about 1,000 nmole/g_(wcm)/hr, about 1,000 to about 5,000nmole/g_(wcm)/hr, about 2,000 to about 20,000 nmole/g_(wcm)/hr, about5,000 to about 50,000 nmole/g_(wcm)/hr, about 10,000 to about 100,000nmole/g_(wcm)/hr, about 20,000 to about 150,000 nmole/g_(wcm)/hr, orabout 20,000 to about 200,000 nmole/g_(wcm)/hr.

The amount of isoprene in units of nmole/g_(wcm)/hr can be measured asdisclosed in U.S. Pat. No. 5,849,970, which is hereby incorporated byreference in its entirety, particularly with respect to the measurementof isoprene production. For example, two mL of headspace (e.g.,headspace from a culture such as 2 mL of culture cultured in sealedvials at 32° C. with shaking at 200 rpm for approximately 3 hours) areanalyzed for isoprene using a standard gas chromatography system, suchas a system operated isothermally (85° C.) with an n-octane/porasil Ccolumn (Alltech Associates, Inc., Deerfield, Ill.) and coupled to a RGD2mercuric oxide reduction gas detector (Trace Analytical, Menlo Park,Calif.) (see, for example, Greenberg et al, Atmos. Environ. 27A:2689-2692, 1993; Silver et al., Plant Physiol. 97:1588-1591, 1991, whichare each hereby incorporated by reference in their entireties,particularly with respect to the measurement of isoprene production).The gas chromatography area units are converted to nmol isoprene via astandard isoprene concentration calibration curve. In some embodiments,the value for the grams of cells for the wet weight of the cells iscalculated by obtaining the A₆₀₀ value for a sample of the cell culture,and then converting the A₆₀₀ value to grams of cells based on acalibration curve of wet weights for cell cultures with a known A₆₀₀value. In some embodiments, the grams of the cells is estimated byassuming that one liter of broth (including cell medium and cells) withan A₆₀₀ value of 1 has a wet cell weight of 1 gram. The value is alsodivided by the number of hours the culture has been incubating for, suchas three hours.

In some embodiments, the cells in culture produce isoprene at greaterthan or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600,700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000,5,000, 10,000, 100,000, or more ng of isoprene/gram of cells for the wetweight of the cells/hr (ng/g_(wcm)/h). In some embodiments, the amountof isoprene is between about 2 to about 5,000 ng/g_(wcm)/h, such asbetween about 2 to about 100 ng/g_(wcm)/h, about 100 to about 500ng/g_(wcm)/h, about 500 to about 1,000 ng/g_(wcm)/h, about 1,000 toabout 2,000 ng/g_(wcm)/h, or about 2,000 to about 5,000 ng/g_(wcm)/h. Insome embodiments, the amount of isoprene is between about 20 to about5,000 ng/g_(wcm)/h, about 100 to about 5,000 ng/g_(wcm)/h, about 200 toabout 2,000 ng/g_(wcm)/h, about 200 to about 1,000 ng/g_(wcm)/h, about300 to about 1,000 ng/g_(wcm)/h, or about 400 to about 1,000ng/g_(wcm)/h. The amount of isoprene in ng/g_(wcm)/h can be calculatedby multiplying the value for isoprene production in the units ofnmole/g_(wcm)/hr discussed above by 68.1 (as described in Equation 5below).

In some embodiments, the cells in culture produce a cumulative titer(total amount) of isoprene at greater than or about 1, 10, 25, 50, 100,150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500,1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000, 50,000, 100,000, ormore mg of isoprene/L of broth (mg/L_(broth), wherein the volume ofbroth includes the volume of the cells and the cell medium). In someembodiments, the amount of isoprene is between about 2 to about 5,000mg/L_(broth), such as between about 2 to about 100 mg/L_(broth), about100 to about 500 mg/L_(broth), about 500 to about 1,000 mg/L_(broth),about 1,000 to about 2,000 mg/L_(broth), or about 2,000 to about 5,000mg/L_(broth). In some embodiments, the amount of isoprene is betweenabout 20 to about 5,000 mg/L_(broth), about 100 to about 5,000mg/L_(broth), about 200 to about 2,000 mg/L_(broth), about 200 to about1,000 mg/L_(broth), about 300 to about 1,000 mg/L_(broth), or about 400to about 1,000 mg/L_(broth).

The specific productivity of isoprene in mg of isoprene/L of headspacefrom shake flask or similar cultures can be measured by taking a 1 mlsample from the cell culture at an OD₆₀₀ value of approximately 1.0,putting it in a 20 mL vial, incubating for 30 minutes, and thenmeasuring the amount of isoprene in the headspace (as described, forexample, in Example I, part II). If the OD₆₀₀ value is not 1.0, then themeasurement can be normalized to an OD₆₀₀ value of 1.0 by dividing bythe OD₆₀₀ value. The value of mg isoprene/L headspace can be convertedto mg/L_(broth)/hr/OD₆₀₀ of culture broth by multiplying by a factor of38. The value in units of mg/L_(broth)/hr/OD₆₀₀ can be multiplied by thenumber of hours and the OD₆₀₀ value to obtain the cumulative titer inunits of mg of isoprene/L of broth.

In some embodiments, the cells in culture have an average volumetricproductivity of isoprene at greater than or about 0.1, 1.0, 10, 25, 50,100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1100,1200, 1300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100,2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100,3,200, 3,300, 3,400, 3,500, or more mg of isoprene/L of broth/hr(mg/L_(broth)/hr, wherein the volume of broth includes the volume of thecells and the cell medium). In some embodiments, the average volumetricproductivity of isoprene is between about 0.1 to about 3,500mg/L_(broth)/hr, such as between about 0.1 to about 100 mg/L_(broth)/hr,about 100 to about 500 mg/L_(broth)/hr, about 500 to about 1,000mg/L_(broth)/hr, about 1,000 to about 1,500 mg/L_(broth)/hr, about 1,500to about 2,000 mg/L_(broth)/hr, about 2,000 to about 2,500mg/L_(broth)/hr, about 2,500 to about 3,000 mg/L_(broth)/hr, or about3,000 to about 3,500 mg/L_(broth)/hr. In some embodiments, the averagevolumetric productivity of isoprene is between about 10 to about 3,500mg/L_(broth)/hr, about 100 to about 3,500 mg/L_(broth)/hr, about 200 toabout 1,000 mg/L_(broth)/hr, about 200 to about 1,500 mg/L_(broth)/hr,about 1,000 to about 3,000 mg/L_(broth)/hr, or about 1,500 to about3,000 mg/L_(broth)/hr.

In some embodiments, the cells in culture have a peak volumetricproductivity of isoprene at greater than or about 0.5, 1.0, 10, 25, 50,100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1100,1200, 1300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100,2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100,3,200, 3,300, 3,400, 3,500, 3,750, 4,000, 4,250, 4,500, 4,750, 5,000,5,250, 5,500, 5,750, 6,000, 6,250, 6,500, 6,750, 7,000, 7,250, 7,500,7,750, 8,000, 8,250, 8,500, 8,750, 9,000, 9,250, 9,500, 9,750, 10,000,12,500, 15,000, or more mg of isoprene/L of broth/hr (mg/L_(broth)/hr,wherein the volume of broth includes the volume of the cells and thecell medium). In some embodiments, the peak volumetric productivity ofisoprene is between about 0.5 to about 15,000 mg/L_(broth)/hr, such asbetween about 0.5 to about 10 mg/L_(broth)/hr, about 1.0 to about 100mg/L_(broth)/hr, about 100 to about 500 mg/L_(broth)/hr, about 500 toabout 1,000 mg/L_(broth)/hr, about 1,000 to about 1,500 mg/L_(broth)/hr,about 1,500 to about 2,000 mg/L_(broth)/hr, about 2,000 to about 2,500mg/L_(broth)/hr, about 2,500 to about 3,000 mg/L_(broth)/hr, about 3,000to about 3,500 mg/L_(broth)/hr, about 3,500 to about 5,000mg/L_(broth)/hr, about 5,000 to about 7,500 mg/L_(broth)/hr, about 7,500to about 10,000 mg/L_(broth)/hr, about 10,000 to about 12,500mg/L_(broth)/h, or about 12,500 to about 15,000 mg/L_(broth)/hr. In someembodiments, the peak volumetric productivity of isoprene is betweenabout 10 to about 15,000 mg/L_(broth)/hr, about 100 to about 2,500mg/L_(broth)/hr, about 1,000 to about 5,000 mg/L_(broth)/hr, about 2,500to about 7,500 mg/L_(broth)/hr, about 5,000 to about 10,000mg/L_(broth)/hr, about 7,500 to about 12,500 mg/L_(broth)/hr, or about10,000 to about 15,000 mg/L_(broth)/hr.

The instantaneous isoprene production rate in mg/L_(broth)/hr in afermentor can be measured by taking a sample of the fermentor off-gas,analyzing it for the amount of isoprene (in units such as mg of isopreneper L_(gas)) as described, for example, in Example I, part II andmultiplying this value by the rate at which off-gas is passed thougheach liter of broth (e.g., at 1 vvm (volume of air/volume ofbroth/minute) this is 60 L_(gas) per hour). Thus, an off-gas level of 1mg/L_(gas) corresponds to an instantaneous production rate of 60mg/L_(broth)/hr at air flow of 1 vvm. If desired, the value in the unitsmg/L_(broth)/hr can be divided by the OD₆₀₀ value to obtain the specificrate in units of mg/L_(broth)/hr/OD. The average value of mgisoprene/L_(gas) can be converted to the total product productivity(grams of isoprene per liter of fermentation broth, mg/L_(broth)) bymultiplying this average off-gas isoprene concentration by the totalamount of off-gas sparged per liter of fermentation broth during thefermentation. Thus, an average off-gas isoprene concentration of 0.5mg/L_(broth)/hr over 10 hours at 1 vvm corresponds to a total productconcentration of 300 mg isoprene/L_(broth).

In some embodiments, the cells in culture convert greater than or about0.0015, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.12, 0.14, 0.16, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 2.0, 2.2, 2.4, 2.6,3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0,16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 23.2, 23.4, 23.6, 23.8,24.0, 25.0, 30.0, 31.0, 32.0, 33.0, 35.0, 37.5, 40.0, 45.0, 47.5, 50.0,55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, or 90.0 molar % of the carbonin the cell culture medium into isoprene. In some embodiments, thepercent conversion of carbon into isoprene is between about 0.002 toabout 90.0 molar %, such as about 0.002 to about 0.005%, about 0.005 toabout 0.01%, about 0.01 to about 0.05%, about 0.05 to about 0.15%, 0.15to about 0.2%, about 0.2 to about 0.3%, about 0.3 to about 0.5%, about0.5 to about 0.8%, about 0.8 to about 1.0%, about 1.0 to about 1.6%,about 1.6 to about 3.0%, about 3.0 to about 5.0%, about 5.0 to about8.0%, about 8.0 to about 10.0%, about 10.0 to about 15.0%, about 15.0 toabout 20.0%, about 20.0 to about 25.0%, about 25.0% to 30.0%, about30.0% to 35.0%, about 35.0% to 40.0%, about 45.0% to 50.0%, about 50.0%to 55.0%, about 55.0% to 60.0%, about 60.0% to 65.0%, about 65.0% to70.0%, about 75.0% to 80.0%, about 80.0% to 85.0%, or about 85.0% to90.0%. In some embodiments, the percent conversion of carbon intoisoprene is between about 0.002 to about 0.4 molar %, 0.002 to about0.16 molar %, 0.04 to about 0.16 molar %, about 0.005 to about 0.3 molar%, about 0.01 to about 0.3 molar %, about 0.05 to about 0.3 molar %,about 0.1 to 0.3 molar %, about 0.3 to about 1.0 molar %, about 1.0 toabout 5.0 molar %, about 2 to about 5.0 molar %, about 5.0 to about 10.0molar %, about 7 to about 10.0 molar %, about 10.0 to about 20.0 molar%, about 12 to about 20.0 molar %, about 16 to about 20.0 molar %, about18 to about 20.0 molar %, about 18 to 23.2 molar %, about 18 to 23.6molar %, about 18 to about 23.8 molar %, about 18 to about 24.0 molar %,about 18 to about 25.0 molar %, about 20 to about 30.0 molar %, about 30to about 40.0 molar %, about 30 to about 50.0 molar %, about 30 to about60.0 molar %, about 30 to about 70.0 molar %, about 30 to about 80.0molar %, or about 30 to about 90.0 molar %

The percent conversion of carbon into isoprene (also referred to as “%carbon yield”) can be measured by dividing the moles carbon in theisoprene produced by the moles carbon in the carbon source (such as themoles of carbon in batched and fed glucose and yeast extract). Thisnumber is multiplied by 100% to give a percentage value (as indicated inEquation 1).% Carbon Yield=(moles carbon in isoprene produced)/(moles carbon incarbon source)*100  Equation 1

For this calculation, yeast extract can be assumed to contain 50% w/wcarbon. As an example, for the 500 liter described in Example 7, partVIII, the percent conversion of carbon into isoprene can be calculatedas shown in Equation 2.Carbon Yield=(39.1 g isoprene*1/68.1 mol/g*5 C/mol)/[(181221 g glucose*1/180 mol/g*6 C/mol)+(17780 g yeast extract*0.5* 1/12mol/g)]*100=0.042%  Equation 2

For the two 500 liter fermentations described herein (Example 7, partsVII and VIII), the percent conversion of carbon into isoprene wasbetween 0.04-0.06%. A 0.11-0.16% carbon yield has been achieved using 14liter systems as described herein.

One skilled in the art can readily convert the rates of isopreneproduction or amount of isoprene produced into any other units.Exemplary equations are listed below for interconverting between units.

Units for Rate of Isoprene Production (Total and Specific)1 g isoprene/L_(broth)/hr=14.7 mmol isoprene/L_(broth)/hr (totalvolumetric rate)  Equation 31 nmol isoprene/g_(wcm)/hr=1 nmol isoprene/L_(broth)/hr/OD₆₀₀  Equation4(This conversion assumes that one liter of broth with an OD₆₀₀ value of1 has a wet cell weight of 1 gram.)1 nmol isoprene/g_(wcm)/hr=68.1 ng isoprene/g_(wcm)/hr  Equation 5(given the molecular weight of isoprene)1 nmol isoprene/L_(gas) O₂/hr=90 nmol isoprene/L_(broth)/hr  Equation 6(at an O₂ flow rate of 90 L/hr per L of culture broth)1 ug isoprene/L_(gas) isoprene in off-gas=60 ug isoprene/L_(broth)/hr ata flow rate of 60 L_(gas) per L_(broth) (1 vvm)  Equation 7Units for Titer (Total and Specific)1 nmol isoprene/mg cell protein=150 nmolisoprene/L_(broth)/OD₆₀₀  Equation 8(This conversion assumes that one liter of broth with an OD₆₀₀ value of1 has a total cell protein of approximately 150 mg) (specificproductivity)1 g isoprene/L_(broth)=14.7 mmol isoprene/L_(broth) (totaltiter)  Equation 9

If desired, Equation 10 can be used to convert any of the units thatinclude the wet weight of the cells into the corresponding units thatinclude the dry weight of the cells.Dry weight of cells=(wet weight of cells)/3.3  Equation 10

In some embodiments encompassed by the invention, a cell comprising aheterologous nucleic acid encoding an isoprene synthase polypeptideproduces an amount of isoprene that is at least or about 2-fold, 3-fold,5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 150-fold, 200-fold,400-fold, or greater than the amount of isoprene produced from acorresponding cell grown under essentially the same conditions withoutthe heterologous nucleic acid encoding the isoprene synthasepolypeptide.

In some embodiments encompassed by the invention, a cell comprising aheterologous nucleic acid encoding an isoprene synthase polypeptide andone or more heterologous nucleic acids encoding a DXS, IDI, and/or MVApathway polypeptide produces an amount of isoprene that is at least orabout 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold,150-fold, 200-fold, 400-fold, or greater than the amount of isopreneproduced from a corresponding cell grown under essentially the sameconditions without the heterologous nucleic acids.

Exemplary Isoprene Purification Methods

In some embodiments, any of the methods described herein further includerecovering the isoprene. For example, the isoprene produced using thecompositions and methods of the invention can be recovered usingstandard techniques. such as gas stripping, fractionation,adsorption/desorption, pervaporation, thermal or vacuum desorption ofisoprene from a solid phase, or extraction of isoprene immobilized orabsorbed to a solid phase with a solvent (see, for example, U.S. Pat.Nos. 4,703,007 and 4,570,029, which are each hereby incorporated byreference in their entireties, particularly with respect to isoprenerecovery and purification methods). In some embodiments, the recovery ofisoprene involves the isolation of isoprene in a liquid form (such as aneat solution of isoprene or a solution of isoprene in a solvent). Gasstripping involves the removal of isoprene vapor from the fermentationoff-gas stream in a continuous manner. Such removal can be achieved inseveral different ways including, but not limited to, adsorption to asolid phase, partition into a liquid phase, or direct condensation. Insome embodiments, membrane enrichment of a dilute isoprene vapor streamabove the dew point of the vapor resulting in the condensation of liquidisoprene.

The recovery of isoprene may involve one step or multiple steps. In someembodiments, the removal of isoprene vapor from the fermentation off-gasand the conversion of isoprene to a liquid phase are performedsimultaneously. For example, isoprene can be directly condensed from theoff-gas stream to form a liquid. In some embodiments, the removal ofisoprene vapor from the fermentation off-gas and the conversion ofisoprene to a liquid phase are performed sequentially. For example,isoprene may be adsorbed to a solid phase and then extracted from thesolid phase with a solvent.

In some embodiments, any of the methods described herein further includepurifying the isoprene. For example, the isoprene produced using thecompositions and methods of the invention can be purified using standardtechniques. Purification refers to a process through which isoprene isseparated from one or more components that are present when the isopreneis produced. In some embodiments, the isoprene is obtained as asubstantially pure liquid. Examples of purification methods include (i)distillation from a solution in a liquid extractant and (ii)chromatography. As used herein, “purified isoprene” means isoprene thathas been separated from one or more components that are present when theisoprene is produced. In some embodiments, the isoprene is at leastabout 20%, by weight, free from other components that are present whenthe isoprene is produced. In various embodiments, the isoprene is atleast or about 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 99%,by weight, pure. Purity can be assayed by any appropriate method, e.g.,by column chromatography, HPLC analysis, or GC-MS analysis.

In some embodiments, any of the methods described herein further includepolymerizing the isoprene. For example, standard methods can be used topolymerize the purified isoprene to form cis-polyisoprene or other downstream products using standard methods.

Additional methods and compositions are described in U.S. Provisionalpatent application No. 61/097,186, filed on Sep. 15, 2008, U.S.Provisional patent application No. 61/097,189, filed on Sep. 15, 2008,and U.S. Provisional patent application No. 61/097,163, filed on Sep.15, 2008, all of which are incorporated by reference in theirentireties, particular with respect to compositions and methods forproducing isoprene.

EXAMPLES

The examples, which are intended to be purely exemplary of the inventionand should therefore not be considered to limit the invention in anyway, also describe and detail aspects and embodiments of the inventiondiscussed above. Unless indicated otherwise, temperature is in degreesCentigrade and pressure is at or near atmospheric. The foregoingexamples and detailed description are offered by way of illustration andnot by way of limitation. All publications, patent applications, andpatents cited in this specification are herein incorporated by referenceas if each individual publication, patent application, or patent werespecifically and individually indicated to be incorporated by reference.In particular, all publications cited herein are expressly incorporatedherein by reference for the purpose of describing and disclosingcompositions and methodologies which might be used in connection withthe invention. Although the foregoing invention has been described insome detail by way of illustration and example for purposes of clarityof understanding, it will be readily apparent to those of ordinary skillin the art in light of the teachings of this invention that certainchanges and modifications may be made thereto without departing from thespirit or scope of the appended claims.

Example 1 Production of Isoprene in E. coli Expressing Recombinant KudzuIsoprene Synthase

I. Construction of Vectors for Expression of the Kudzu Isoprene Synthasein E. coli

The protein sequence for the kudzu (Pueraria montana) isoprene synthasegene (IspS) was obtained from GenBank (AAQ84170). A kudzu isoprenesynthase gene, optimized for E. coli codon usage, was purchased fromDNA2.0 (SEQ ID NO:1). The isoprene synthase gene was removed from thesupplied plasmid by restriction endonuclease digestion withBspLU11I/PstI, gel-purified, and ligated into pTrcHis2B (Invitrogen)that had been digested with NcoI/PstI. The construct was designed suchthat the stop codon in the isoprene synthase gene 5′ to the PstI site.As a result, when the construct was expressed the His-Tag is notattached to the isoprene synthase protein. The resulting plasmid,pTrcKudzu, was verified by sequencing (FIGS. 2 and 3).

The isoprene synthase gene was also cloned into pET16b (Novagen). Inthis case, the isoprene synthase gene was inserted into pET16b such thatthe recombinant isoprene synthase protein contained the N-terminal Histag. The isoprene synthase gene was amplified from pTrcKudzu by PCRusing the primer set pET-His-Kudzu-2F:5′-CGTGAGATCATATGTGTGCGACCTCTTCTCAATTTAC (SEQ ID NO:3) andpET-His-Kudzu-R: 5′-CGGTCGACGGATCCCTGCAGTTAGACATACATCAGCTG (SEQ IDNO:4). These primers added an NdeI site at the 5′-end and a BamH1 siteat the 3′ end of the gene respectively. The plasmid pTrcKudzu, describedabove, was used as template DNA, Herculase polymerase (Stratagene) wasused according to manufacture's directions, and primers were added at aconcentration of 10 pMols. The PCR was carried out in a total volume of25 μl. The PCR product was digested with NdeI/BamH1 and cloned intopET16b digested with the same enzymes. The ligation mix was transformedinto E. coli Top10 (Invitrogen) and the correct clone selected bysequencing. The resulting plasmid, in which the kudzu isoprene synthasegene was expressed from the T7 promoter, was designated pETNHisKudzu(FIGS. 4 and 5).

The kudzu isoprene synthase gene was also cloned into the low copynumber plasmid pCL1920. Primers were used to amplify the kudzu isoprenesynthase gene from pTrcKudzu described above. The forward primer added aHindIII site and an E. coli consensus RBS to the 5′ end. The PstIcloning site was already present in pTrcKudzu just 3′ of the stop codonso the reverse primer was constructed such that the final PCR productincludes the PstI site. The sequences of the primers were:HindIII-rbs-Kudzu F: 5′-CATATGAAAGCTTGTATCGATTAAATAAGGAGGAATAAACC (SEQID NO:6) and BamH1-Kudzu R:

5′-CGGTCGACGGATCCCTGCAGTTAGACATACATCAGCTG (SEQ ID NO:4). The PCR productwas amplified using Herculase polymerase with primers at a concentrationof 10 pmol and with 1 ng of template DNA (pTrcKudzu). The amplificationprotocol included 30 cycles of (95° C. for 1 minute, 60° C. for 1minute, 72° C. for 2 minutes). The product was digested with HindIII andPstI and ligated into pCL1920 which had also been digested with HindIIIand PstI. The ligation mix was transformed into E. coli Top10. Severaltransformants were checked by sequencing. The resulting plasmid wasdesignated pCL-lac-Kudzu (FIGS. 6 and 7).

II. Determination of Isoprene Production

For the shake flask cultures, one ml of a culture was transferred fromshake flasks to 20 ml CTC headspace vials (Agilent vial cat #5188 2753;cap cat #5188 2759). The cap was screwed on tightly and the vialsincubated at the equivalent temperature with shaking at 250 rpm. After30 minutes the vials were removed from the incubator and analyzed asdescribed below (see Table 1 for some experimental values from thisassay).

In cases where isoprene production in fermentors was determined, sampleswere taken from the off-gas of the fermentor and analyzed directly asdescribed below (see Table 2 for some experimental values from thisassay).

The analysis was performed using an Agilent 6890 GC/MS system interfacedwith a CTC Analytics (Switzerland) CombiPAL autosampler operating inheadspace mode. An Agilent HP-5MS GC/MS column (30 m×0.25 mm; 0.25 μmfilm thickness) was used for separation of analytes. The sampler was setup to inject 500 μL of headspace gas. The GC/MS method utilized heliumas the carrier gas at a flow of 1 ml/minutes. The injection port washeld at 250° C. with a split ratio of 50:1. The oven temperature washeld at 37° C. for the 2 minute duration of the analysis. The Agilent5793N mass selective detector was run in single ion monitoring (SIM)mode on m/z 67. The detector was switched off from 1.4 to 1.7 minutes toallow the elution of permanent gases. Under these conditions isoprene(2-methyl-1,3-butadiene) was observed to elute at 1.78 minutes. Acalibration table was used to quantify the absolute amount of isopreneand was found to be linear from 1 μg/L to 200 μg/L. The limit ofdetection was estimated to be 50 to 100 ng/L using this method.

III. Production of Isoprene in Shake Flasks Containing E. coli CellsExpressing Recombinant Isoprene Synthase

The vectors described above were introduced to E. coli strain BL21(Novagen) to produce strains BL21/ptrcKudzu, BL21/pCL-lac-Kudzu andBL21/pETHisKudzu. The strains were spread for isolation onto LA (Luriaagar) and carbenicillin (50 μg/ml) and incubated overnight at 37° C.Single colonies were inoculated into 250 ml baffled shake flaskscontaining 20 ml Luria Bertani broth (LB) and carbenicillin (100 μg/ml).Cultures were grown overnight at 20° C. with shaking at 200 rpm. TheOD₆₀₀ of the overnight cultures were measured and the cultures werediluted into a 250 ml baffled shake flask containing 30 ml MagicMedia(Invitrogen) and carbenicillin (100 μg/ml) to an OD₆₀₀˜0.05. The culturewas incubated at 30° C. with shaking at 200 rpm. When the OD₆₀₀˜0.5-0.8,400 μM IPTG was added and the cells were incubated for a further 6 hoursat 30° C. with shaking at 200 rpm. At 0, 2, 4 and 6 hours afterinduction with IPTG, 1 ml aliquots of the cultures were collected, theOD₆₀₀ was determined and the amount of isoprene produced was measured asdescribed above. Results are shown in FIG. 8.

IV. Production of Isoprene from BL21/ptrcKudzu in 14 Liter Fermentation

Large scale production of isoprene from E. coli containing therecombinant kudzu isoprene synthase gene was determined from a fed-batchculture. The recipe for the fermentation media (TM2) per liter offermentation medium was as follows: K₂HPO₄ 13.6 g, KH₂PO₄ 13.6 g,MgSO4*7H₂O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3g, (NH₄)₂SO₄ 3.2 g, yeast extract 5 g, 1000× Modified Trace MetalSolution 1 ml. All of the components were added together and dissolvedin diH₂O. The pH was adjusted to 6.8 with potassium hydroxide (KOH) andq.s. to volume. The final product was filter sterilized with 0.22μfilter (only, do not autoclave). The recipe for 1000× Modified TraceMetal Solution was as follows: Citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g,NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100mg, H₃BO₃ 100 mg, NaMoO₄*2H₂O 100 mg. Each component was dissolved oneat a time in diH₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume andfilter sterilized with a 0.22μ filter.

This experiment was carried out in 14 L bioreactor to monitor isopreneformation from glucose at the desired fermentation, pH 6.7 andtemperature 34° C. An inoculum of E. coli strain BL21/ptrcKudzu takenfrom a frozen vial was prepared in soytone-yeast extract-glucose medium.After the inoculum grew to OD₅₅₀=0.6, two 600 ml flasks were centrifugedand the contents resuspended in 70 ml supernatant to transfer the cellpellet (70 ml of OD 3.1 material) to the bioreactor. At various timesafter inoculation, samples were removed and the amount of isopreneproduced was determined as described above. Results are shown in FIG. 9.

Example 2 Production of Isoprene in E. coli Expressing RecombinantPoplar Isoprene Synthase

The protein sequence for the poplar (Populus alba×Populus tremula)isoprene synthase (Schnitzler, J-P, et al. (2005) Planta 222:777-786)was obtained from GenBank (CAC35696). A gene, codon optimized for E.coli, was purchased from DNA2.0 (p9796-poplar, FIGS. 30 and 31). Theisoprene synthase gene was removed from the supplied plasmid byrestriction endonuclease digestion with BspLU11I/PstI, gel-purified, andligated into pTrcHis2B that had been digested with NcoI/PstI. Theconstruct is cloned such that the stop codon in the insert is before thePstI site, which results in a construct in which the His-Tag is notattached to the isoprene synthase protein. The resulting plasmidpTrcPoplar (FIGS. 32 and 33), was verified by sequencing.

Example 3 Production of Isoprene in Panteoa citrea ExpressingRecombinant Kudzu Isoprene Synthase

The pTrcKudzu and pCL-lac Kudzu plasmids described in Example 1 wereelectroporated into P. citrea (U.S. Pat. No. 7,241,587). Transformantswere selected on LA containing carbenicillin (200 μg/ml) orspectinomycin (50 μg/ml) respectively. Production of isoprene from shakeflasks and determination of the amount of isoprene produced wasperformed as described in Example 1 for E. coli strains expressingrecombinant kudzu isoprene synthase. Results are shown in FIG. 10.

Example 4 Production of Isoprene in Bacillus subtilis ExpressingRecombinant Kudzu Isoprene Synthase

I. Construction of a B. subtilis Replicating Plasmid for the Expressionof Kudzu Isoprene Synthase

The kudzu isoprene synthase gene was expressed in Bacillus subtilisaprEnprE Pxyl-comK strain (BG3594comK) using a replicating plasmid(pBS19 with a chloramphenicol resistance cassette) under control of theaprE promoter. The isoprene synthase gene, the aprE promoter and thetranscription terminator were amplified separately and fused using PCR.The construct was then cloned into pBS19 and transformed into B.subtilis.

a) Amplification of the aprE Promoter

The aprE promoter was amplified from chromosomal DNA from Bacillussubtilis using the following primers:

CF 797 (+) Start aprE promoter MfeI (SEQ ID NO: 58)5′-GACATCAATTGCTCCATTTTCTTCTGCTATCCF 07-43 (−) Fuse aprE promoter to Kudzu ispS (SEQ ID NO: 59)5′-ATTGAGAAGAGGTCGCACACACTCTTTACCCTCTCCTTTTAb) Amplification of the Isoprene Synthase Gene

The kudzu isoprene synthase gene was amplified from plasmid pTrcKudzu(SEQ ID NO:2). The gene had been codon optimized for E. coli andsynthesized by DNA 2.0. The following primers were used:

CF 07-42 (+) Fuse the aprE promoter to kudzuisoprene synthase gene (GTG start codon) (SEQ ID NO: 60)5′-TAAAAGGAGAGGGTAAAGAGTGTGTGCGACCTCTTCTCAAT CF 07-45 (−) Fuse the 3′end of kudzu isoprene synthase gene to the terminator (SEQ ID NO: 61)5′-CCAAGGCCGGTTTTTTTTAGACATACATCAGCTGGTTAATCc) Amplification of the Transcription Terminator

The terminator from the alkaline serine protease of Bacillusamyliquefaciens was amplified from a previously sequenced plasmidpJHPms382 using the following primers:

CF 07-44 (+) Fuse the 3′ end of kudzu isoprenesynthase to the terminator (SEQ ID NO: 62)5′-GATTAACCAGCTGATGTATGTCTAAAAAAAACCGGCCTTGGCF 07-46 (−) End of B. amyliquefaciens terminator (BamHI)(SEQ ID NO: 63) 5′-GACATGACGGATCCGATTACGAATGCCGTCTC

The kudzu fragment was fused to the terminator fragment using PCR withthe following primers:

CF 07-42 (+) Fuse the aprE promoter to kudzuisoprene synthase gene (GTG start codon) (SEQ ID NO: 61)5′-TAAAAGGAGAGGGTAAAGAGTGTGTGCGACCTCTTCTCAATCF 07-46 (−) End of B. amyliquefaciens terminator (BamHI)(SEQ ID NO: 63) 5′-GACATGACGGATCCGATTACGAATGCCGTCTC

The kudzu-terminator fragment was fused to the promoter fragment usingPCR with the following primers:

CF 797 (+) Start aprE promoter MfeI (SEQ ID NO: 64)5′-GACATCAATTGCTCCATTTTCTTCTGCTATCCF 07-46 (−) End of B. amyliquefaciens terminator (BamHI)(SEQ ID NO: 63) 5′-GACATGACGGATCCGATTACGAATGCCGTCTC

The fusion PCR fragment was purified using a Qiagen kit and digestedwith the restriction enzymes MfeI and BamHI. This digested DNA fragmentwas gel purified using a Qiagen kit and ligated to a vector known aspBS19, which had been digested with EcoRI and BamHI and gel purified.

The ligation mix was transformed into E. coli Top 10 cells and colonieswere selected on LA and 50 carbenicillin plates. A total of six colonieswere chosen and grown overnight in LB and 50 carbenicillin and thenplasmids were isolated using a Qiagen kit. The plasmids were digestedwith EcoRI and BamHI to check for inserts and three of the correctplasmids were sent in for sequencing with the following primers:

CF 149 (+) EcoRI start of aprE promoter (SEQ ID NO: 65)5′-GACATGAATTCCTCCATTTTCTTCTGCCF 847 (+) Sequence in pXX 049 (end of aprE promoter) (SEQ ID NO: 66)5′-AGGAGAGGGTAAAGAGTGAG CF 07-45 (−) Fuse the 3′ end of kudzu isoprenesynthase to the terminator (SEQ ID NO: 61)5′-CCAAGGCCGGTTTTTTTTAGACATACATCAGCTGGTTAATCCF 07-48 (+) Sequencing primer for kudzu isoprene synthase(SEQ ID NO: 67) 5′-CTTTTCCATCACCCACCTGAAGCF 07-49 (+) Sequencing in kudzu isoprene synthase (SEQ ID NO: 68)5′-GGCGAAATGGTCCAACAACAAAATTATC

The plasmid designated pBS Kudzu #2 (FIGS. 52 and 12) was correct bysequencing and was transformed into BG 3594 comK, a Bacillus subtilishost strain. Selection was done on LA and 5 chloramphenicol plates. Atransformant was chosen and struck to single colonies on LA and 5chloramphenicol, then grown in LB and 5 chloramphenicol until it reachedan OD₆₀₀ of 1.5. It was stored frozen in a vial at −80° C. in thepresence of glycerol. The resulting strain was designated CF 443.

II. Production of Isoprene in Shake Flasks Containing B. subtilis CellsExpressing Recombinant Isoprene Synthase

Overnight cultures were inoculated with a single colony of CF 443 from aLA and Chloramphenicol (Cm, 25 μg/ml). Cultures were grown in LB and Cmat 37° C. with shaking at 200 rpm. These overnight cultures (1 ml) wereused to inoculate 250 ml baffled shake flasks containing 25 ml Grants IImedia and chloramphenicol at a final concentration of 25 μg/ml. GrantsII Media recipe was 10 g soytone, 3 ml 1M K₂HPO₄, 75 g glucose, 3.6 gurea, 100 ml 10×MOPS, q.s. to 1 L with H₂O, pH 7.2; 10×MOPS recipe was83.72 g MOPS, 7.17 g tricine, 12 g KOH pellets, 10 ml 0.276M K₂SO₄solution, 10 ml 0.528M MgCl₂ solution, 29.22 g NaCl, 100 ml 100×micronutrients, q.s. to 1 L with H₂O; and 100× micronutrients recipe was1.47 g CaCl₂*2H₂O, 0.4 g FeSO₄*7H₂O, 0.1 g MnSO₄*H₂O, 0.1 g ZnSO₄*H₂O,0.05 g CuCl₂*2H₂O, 0.1 g CoCl₂*6H₂O, 0.1 g Na₂MoO₄*2H₂O, q.s. to 1 Lwith H₂O, Shake flasks were incubated at 37° C. and samples were takenat 18, 24, and 44 hours. At 18 hours the headspaces of CF443 and thecontrol strain were sampled. This represented 18 hours of accumulationof isoprene. The amount of isoprene was determined by gas chromatographyas described in Example 1. Production of isoprene was enhancedsignificantly by expressing recombinant isoprene synthase (FIG. 11).

III. Production of Isoprene by CF443 in 14 L Fermentation

Large scale production of isoprene from B. subtilis containing therecombinant kudzu isoprene synthase gene on a replication plasmid wasdetermined from a fed-batch culture. Bacillus strain CF 443, expressinga kudzu isoprene synthase gene, or control stain which does not expressa kudzu isoprene synthase gene were cultivated by conventional fed-batchfermentation in a nutrient medium containing soy meal (Cargill), sodiumand potassium phosphate, magnesium sulfate and a solution of citricacid, ferric chloride and manganese chloride. Prior to fermentation themedia is macerated for 90 minutes using a mixture of enzymes includingcellulases, hemicellulases and pectinases (see, WO95/04134). 14-L batchfermentations are fed with 60% wt/wt glucose (Cargill DE99 dextrose, ADMVersadex greens or Danisco invert sugar) and 99% wt/wt oil (WesternFamily soy oil, where the 99% wt/wt is the concentration of oil beforeit was added to the cell culture medium). Feed was started when glucosein the batch was non-detectable. The feed rate was ramped over severalhours and was adjusted to add oil on an equal carbon basis. The pH wascontrolled at 6.8-7.4 using 28% w/v ammonium hydroxide. In case offoaming, antifoam agent was added to the media. The fermentationtemperature was controlled at 37° C. and the fermentation culture wasagitated at 750 rpm. Various other parameters such as pH, DO %, airflow,and pressure were monitored throughout the entire process. The DO % ismaintained above 20. Samples were taken over the time course of 36 hoursand analyzed for cell growth (OD₅₅₀) and isoprene production. Results ofthese experiments are presented in FIGS. 53A and 53B.

IV. Integration of the Kudzu Isoprene Synthase (ispS) in B. subtilis.

The kudzu isoprene synthase gene was cloned in an integrating plasmid(pJH101-cmpR) under the control of the aprE promoter. Under theconditions tested, no isoprene was detected.

Example 5 Production of Isoprene in Trichoderma

I. Construction of Vectors for Expression of the Kudzu Isoprene Synthasein Trichoderma reesei

The Yarrowia lipolytica codon-optimized kudzu IS gene was synthesized byDNA 2.0 (SEQ ID NO:8) (FIG. 13). This plasmid served as the template forthe following PCR amplification reaction: 1 pJ plasmid template (20ng/ul), 1 μl Primer EL-945 (10 uM)5′-GCTTATGGATCCTCTAGACTATTACACGTACATCAATTGG (SEQ ID NO:9), 1 μl PrimerEL-965 (10 uM) 5′-CACCATGTGTGCAACCTCCTCCCAGTTTAC (SEQ ID NO:10), 1 μldNTP (10 mM), 5 μl 10× PfuUltra II Fusion HS DNA Polymerase Buffer, 1 μlPfuUltra II Fusion HS DNA Polymerase, 40 μl water in a total reactionvolume of 50 μl. The forward primer contained an additional 4nucleotides at the 5′-end that did not correspond to the Y. lipolyticacodon-optimized kudzu isoprene synthase gene, but was required forcloning into the pENTR/D-TOPO vector. The reverse primer contained anadditional 21 nucleotides at the 5′-end that did not correspond to theY. lipolytica codon-optimized kudzu isoprene synthase gene, but wereinserted for cloning into other vector backbones. Using the MJ ResearchPTC-200 Thermocycler, the PCR reaction was performed as follows: 95° C.for 2 minutes (first cycle only), 95° C. for 30 seconds, 55° C. for 30seconds, 72° C. for 30 seconds (repeat for 27 cycles), 72° C. for 1minute after the last cycle. The PCR product was analyzed on a 1.2%E-gel to confirm successful amplification of the Y. lipolyticacodon-optimized kudzu isoprene synthase gene.

The PCR product was then cloned using the TOPO pENTR/D-TOPO Cloning Kitfollowing manufacturer's protocol: 1 μl PCR reaction, 1 μl Saltsolution, 1 μl TOPO pENTR/D-TOPO vector and 3 μl water in a totalreaction volume of 6 μl. The reaction was incubated at room temperaturefor 5 minutes. One microliter of TOPO reaction was transformed intoTOP10 chemically competent E. coli cells. The transformants wereselected on LA and 50 μg/ml kanamycin plates. Several colonies werepicked and each was inoculated into a 5 ml tube containing LB and 50μg/ml kanamycin and the cultures grown overnight at 37° C. with shakingat 200 rpm. Plasmids were isolated from the overnight culture tubesusing QIAprep Spin Miniprep Kit, following manufacturer's protocol.Several plasmids were sequenced to verify that the DNA sequence wascorrect.

A single pENTR/D-TOPO plasmid, encoding a Y. lipolytica codon-optimizedkudzu isoprene synthase gene, was used for Gateway Cloning into acustom-made pTrex3g vector. Construction of pTrex3g is described in WO2005/001036 A2. The reaction was performed following manufacturer'sprotocol for the Gateway LR Clonase II Enzyme Mix Kit (Invitrogen): 1 μlY. lipolytica codon-optimized kudzu isoprene synthase gene pENTR/D-TOPOdonor vector, 1 μl pTrex3g destination vector, 6 μl TE buffer, pH 8.0 ina total reaction volume of 8 μl. The reaction was incubated at roomtemperature for 1 hour and then 1 μl proteinase K solution was added andthe incubation continued at 37° C. for 10 minutes. Then 1 μl of reactionwas transformed into TOP10 chemically competent E. coli cells. Thetransformants were selected on LA and 50 μg/ml carbenicillin plates.Several colonies were picked and each was inoculated into a 5 ml tubecontaining LB and 50 μg/ml carbenicillin and the cultures were grownovernight at 37° C. with shaking at 200 rpm. Plasmids were isolated fromthe overnight culture tubes using QIAprep Spin Miniprep Kit (Qiagen,Inc.), following manufacturer's protocol. Several plasmids weresequenced to verify that the DNA sequence was correct.

Biolistic transformation of Y. lipolytica codon-optimized kudzu isoprenesynthase pTrex3g plasmid (FIG. 14) into a quad delete Trichoderma reeseistrain was performed using the Biolistic PDS-1000/HE Particle DeliverySystem (see WO 2005/001036 A2). Isolation of stable transformants andshake flask evaluation was performed using protocol listed in Example 11of patent publication WO 2005/001036 A2.

II. Production of Isoprene in Recombinant Strains of T. reesei

One ml of 15 and 36 hour old cultures of isoprene synthase transformantsdescribed above were transferred to head space vials. The vials weresealed and incubated for 5 hours at 30° C. Head space gas was measuredand isoprene was identified by the method described in Example 1. Two ofthe transformants showed traces of isoprene. The amount of isoprenecould be increased by a 14 hour incubation. The two positive samplesshowed isoprene at levels of about 0.5 μg/L for the 14 hour incubation.The untransformed control showed no detectable levels of isoprene. Thisexperiment shows that T. reesei is capable of producing isoprene fromendogenous precursor when supplied with an exogenous isoprene synthase.

Example 6 Production of Isoprene in Yarrowia

I. Construction of Vectors for Expression of the Kudzu Isoprene Synthasein Yarrowia lipolytica.

The starting point for the construction of vectors for the expression ofthe kudzu isoprene synthase gene in Yarrowia lipolytica was the vectorpSPZ1(MAP29Spb). The complete sequence of this vector (SEQ ID No:11) isshown in FIG. 15.

The following fragments were amplified by PCR using chromosomal DNA of aY. lipolytica strain GICC 120285 as the template: a promotorless form ofthe URA3 gene, a fragment of 18S ribosomal RNA gene, a transcriptionterminator of the Y. lipolytica XPR2 gene and two DNA fragmentscontaining the promoters of XPR2 and ICL1 genes. The following PCRprimers were used:

ICL1 3 (SEQ ID NO: 69) 5′-GGTGAATTCAGTCTACTGGGGATTCCCAAATCTATATATACTGCAGGTGAC ICL1 5 (SEQ ID NO: 70) 5′-GCAGGTGGGAAACTATGCACTCC XPR 3(SEQ ID NO: 71) 5′-CCTGAATTCTGTTGGATTGGAGGATTGGATAGTGGG XPR 5(SEQ ID NO: 72) 5′-GGTGTCGACGTACGGTCGAGCTTATTGACC XPRT3 (SEQ ID NO: 73)5′-GGTGGGCCCGCATTTTGCCACCTACAAGCCAG XPRT 5 (SEQ ID NO: 74)5′-GGTGAATTCTAGAGGATCCCAACGCTGTTGCCTACAACGG Y18S3 (SEQ ID NO: 75)5′-GGTGCGGCCGCTGTCTGGACCTGGTGAGTTTCCCCG Y18S 5 (SEQ ID NO: 76)5′-GGTGGGCCCATTAAATCAGTTATCGTTTATTTGATAG YURA3 (SEQ ID NO: 77)5′-GGTGACCAGCAAGTCCATGGGTGGTTTGATCATGG YURA 50 (SEQ ID NO: 78)5′-GGTGCGGCCGCCTTTGGAGTACGACTCCAACTATG YURA 51 (SEQ ID NO: 79)5′-GCGGCCGCAGACTAAATTTATTTCAGTCTCC

For PCR amplification the PfuUltraII polymerase (Stratagene),supplier-provided buffer and dNTPs, 2.5 μM primers and the indicatedtemplate DNA were used as per the manufacturer's instructions. Theamplification was done using the following cycle: 95° C. for 1 min; 34×(95° C. for 30 sec; 55° C. for 30 sec; 72° C. for 3 min) and 10 min at72° C. followed by a 4° C. incubation.

Synthetic DNA molecules encoding the kudzu isoprene synthase gene,codon-optimized for expression in Yarrowia, was obtained from DNA 2.0(FIG. 16; SEQ ID NO:12). Full detail of the construction scheme of theplasmids pYLA(KZ1) and pYLI(KZ1) carrying the synthetic kudzu isoprenesynthase gene under control of XPR2 and ICL1 promoters respectively ispresented in FIG. 18. Control plasmids in which a mating factor gene(MAP29) is inserted in place of an isoprene synthase gene were alsoconstructed (FIGS. 18E and 18F).

A similar cloning procedure can be used to express a poplar (Populusalba×Populus tremula) isoprene synthase gene. The sequence of the poplarisoprene is described in Miller B. et al. (2001) Planta 213, 483-487 andshown in FIG. 17 (SEQ ID NO:13). A construction scheme for thegeneration the plasmids pYLA(POP1) and pYLI(POP1) carrying syntheticpoplar isoprene synthase gene under control of XPR2 and ICL1 promotersrespectively is presented in FIGS. 18A and B.

II. Production of Isoprene by Recombinant Strains of Y. lipolytica.

Vectors pYLA(KZ1), pYLI(KZ1), pYLA(MAP29) and pYLI(MAP29) were digestedwith SacII and used to transform the strain Y. lipolytica CLIB 122 by astandard lithium acetate/polyethylene glycol procedure to uridineprototrophy. Briefly, the yeast cells grown in YEPD (1% yeast extract,2% peptone, 2% glucose) overnight, were collected by centrifugation(4000 rpm, 10 min), washed once with sterile water and suspended in 0.1M lithium acetate, pH 6.0. Two hundred μl aliquots of the cellsuspension were mixed with linearized plasmid DNA solution (10-20 μg),incubated for 10 minutes at room temperature and mixed with 1 ml of 50%PEG 4000 in the same buffer. The suspensions were further incubated for1 hour at room temperature followed by a 2 minutes heat shock at 42° C.Cells were then plated on SC his leu plates (0.67% yeast nitrogen base,2% glucose, 100 mg/L each of leucine and histidine). Transformantsappeared after 3-4 days of incubation at 30° C.

Three isolates from the pYLA(KZ1) transformation, three isolates fromthe pYLI(KZ1) transformation, two isolates from the pYLA(MAP29)transformation and two isolates from the pYLI(MAP29) transformation weregrown for 24 hours in YEP7 medium (1% yeast extract, 2% peptone, pH 7.0)at 30° C. with shaking. Cells from 10 ml of culture were collected bycentrifugation, resuspended in 3 ml of fresh YEP7 and placed into 15 mlscrew cap vials. The vials were incubated overnight at room temperaturewith gentle (60 rpm) shaking. Isoprene content in the headspace of thesevials was analyzed by gas chromatography using mass-spectrometricdetector as described in Example 1. All transformants obtained withpYLA(KZ1) and pYLI(KZ1) produced readily detectable amounts of isoprene(0.5 μg/L to 1 μg/L, FIG. 20). No isoprene was detected in the headspaceof the control strains carrying phytase gene instead of an isoprenesynthase gene.

Example 7 Production of Isoprene in E. coli Expressing Kudzu IsopreneSynthase and idi, or dxs, or idi and dxs

I. Construction of Vectors Encoding Kudzu Isoprene Synthase and idi, ordxs, or idi and dxs for the Production of Isoprene in E. coli

i) Construction of pTrcKudzuKan

The bla gene of pTrcKudzu (described in Example 1) was replaced with thegene conferring kanamycin resistance. To remove the bla gene, pTrcKudzuwas digested with BspHI, treated with Shrimp Alkaline Phosphatase (SAP),heat killed at 65° C., then end-filled with Klenow fragment and dNTPs.The 5 kbp large fragment was purified from an agarose gel and ligated tothe kan^(r) gene which had been PCR amplified from pCR-Blunt-II-TOPOusing primers MCM22 5′-GATCAAGCTTAACCGGAATTGCCAGCTG (SEQ ID NO:14) andMCM23 5′-GATCCGATCGTCAGAAGAACTCGTCAAGAAGGC (SEQ ID NO:15), digested withHindIII and PvuI, and end-filled. A transformant carrying a plasmidconferring kanamycin resistance (pTrcKudzuKan) was selected on LAcontaining kanamycin 50 μg/ml.

ii) Construction of pTrcKudzu yIDI Kan

pTrcKudzuKan was digested with PstI, treated with SAP, heat killed andgel purified. It was ligated to a PCR product encoding idi from S.cerevisiae with a synthetic RBS. The primers for PCR were NsiI-YIDI 1 F5′-CATCAATGCATCGCCCTTAGGAGGTAAAAAAAAATGAC (SEQ ID NO:16) and PstI-YIDI 1R 5′-CCTTCTGCAGGACGCGTTGTTATAGC (SEQ ID NO:17); and the template was S.cerevisiae genomic DNA. The PCR product was digested with NsiI and PstIand gel purified prior to ligation. The ligation mixture was transformedinto chemically competent TOP10 cells and selected on LA containing 50μg/ml kanamycin. Several transformants were isolated and sequenced andthe resulting plasmid was called pTrcKudzu-yIDI(kan) (FIGS. 34 and 35).

iii) Construction of pTrcKudzu DXS Kan

Plasmid pTrcKudzuKan was digested with PstI, treated with SAP, heatkilled and gel purified. It was ligated to a PCR product encoding dxsfrom E. coli with a synthetic RBS. The primers for PCR were MCM 135′-GATCATGCATTCGCCCTTAGGAGGTAAAAAAACATGAGTTTTGATATTGCCAAATACCCG (SEQ IDNO:18) and MCM14 5′-CATGCTGCAGTTATGCCAGCCAGGCCTTGAT (SEQ ID NO:19); andthe template was E. coli genomic DNA. The PCR product was digested withNsiI and PstI and gel purified prior to ligation. The resultingtransformation reaction was transformed into TOP10 cells and selected onLA with kanamycin 50 μg/ml. Several transformants were isolated andsequenced and the resulting plasmid was called pTrcKudzu-DXS(kan) (FIGS.36 and 37).

iv) Construction of pTrcKudzu-yIDI-dxs (kan)

pTrcKudzu-yIDI(kan) was digested with PstI, treated with SAP, heatkilled and gel purified. It was ligated to a PCR product encoding E.coli dxs with a synthetic RBS (primers MCM135′-GATCATGCATTCGCCCTTAGGAGGTAAAAAAACATGAGTTTTGATATTGCCAAATACCCG (SEQ IDNO:18) and MCM14 5′-CATGCTGCAGTTATGCCAGCCAGGCCTTGAT (SEQ ID NO:19);template TOP10 cells) which had been digested with NsiI and PstI and gelpurified. The final plasmid was called pTrcKudzu-yIDI-dxs (kan) (FIGS.21 and 22).

v) Construction of pCL PtrcKudzu

A fragment of DNA containing the promoter, structural gene andterminator from Example 1 above was digested from pTrcKudzu using SspIand gel purified. It was ligated to pCL1920 which had been digested withPvuII, treated with SAP and heat killed. The resulting ligation mixturewas transformed into TOP10 cells and selected in LA containingspectinomycin 50 μg/ml. Several clones were isolated and sequenced andtwo were selected. pCL PtrcKudzu and pCL PtrcKudzu (A3) have the insertin opposite orientations (FIGS. 38-41).

vi) Construction of pCL PtrcKudzu yIDI

The NsiI-PstI digested, gel purified, IDI PCR amplicon from (ii) abovewas ligated into pCL PtrcKudzu which had been digested with PstI,treated with SAP, and heat killed. The ligation mixture was transformedinto TOP10 cells and selected in LA containing spectinomycin 50 μg/ml.Several clones were isolated and sequenced and the resulting plasmid iscalled pCL PtrcKudzu yIDI (FIGS. 42 and 43).

vii) Construction of pCL PtrcKudzu DXS

The NsiI-PstI digested, gel purified, DXS PCR amplicon from (iii) abovewas ligated into pCL PtrcKudzu (A3) which had been digested with PstI,treated with SAP, and heat killed. The ligation mixture was transformedinto TOP10 cells and selected in LA containing spectinomycin 50 μg/ml.Several clones were isolated and sequenced and the resulting plasmid iscalled pCL PtrcKudzu DXS (FIGS. 44 and 45).

II. Measurement of Isoprene in Headspace from Cultures Expressing KudzuIsoprene Synthase, idi, and/or dxs at Different Copy Numbers.

Cultures of E. coli BL21(λDE3) previously transformed with plasmidspTrcKudzu(kan) (A), pTrcKudzu-yIDI kan (B), pTrcKudzu-DXS kan (C),pTrcKudzu-yIDI-DXS kan (D) were grown in LB kanamycin 50 μg/mL. Culturesof pCL PtrcKudzu (E), pCL PtrcKudzu, pCL PtrcKudzu-yIDI (F) and pCLPtrcKudzu-DXS (G) were grown in LB spectinomycin 50 μg/mL. Cultures wereinduced with 400 μM IPTG at time 0 (OD₆₀₀ approximately 0.5) and samplestaken for isoprene headspace measurement (see Example 1). Results areshown in FIG. 23A-23G.

Plasmid pTrcKudzu-yIDI-dxs (kan) was introduced into E. coli strain BL21by transformation. The resulting strain BL21/pTrc Kudzu IDI DXS wasgrown overnight in LB containing kanamycin (50 μg/ml) at 20° C. and usedto inoculate shake flasks of TM3 (13.6 g K₂PO₄, 13.6 g KH₂PO₄, 2.0 gMgSO₄*7H₂O), 2.0 g citric acid monohydrate, 0.3 g ferric ammoniumcitrate, 3.2 g (NH₄)₂SO₄, 0.2 g yeast extract, 1.0 ml 1000× ModifiedTrace Metal Solution, adjusted to pH 6.8 and q.s. to H₂O, and filtersterilized) containing 1% glucose. Flasks were incubated at 30° C. untilan OD₆₀₀ of 0.8 was reached, and then induced with 400 μM IPTG. Sampleswere taken at various times after induction and the amount of isoprenein the head space was measured as described in Example 1. Results areshown in FIG. 23H.

III. Production of Isoprene from Biomass in E. coli/pTrcKudzu yIDI DXS

The strain BL21 pTrcKudzuIDIDXS was tested for the ability to generateisoprene from three types of biomass; bagasse, corn stover and soft woodpulp with glucose as a control. Hydrolysates of the biomass wereprepared by enzymatic hydrolysis (Brown, L. and Torget, R., 1996, NRELstandard assay method Lap-009 “Enzymatic Saccharification ofLignocellulosic Biomass”) and used at a dilution based upon glucoseequivalents. In this example, glucose equivalents were equal to 1%glucose. A single colony from a plate freshly transformed cells of BL21(DE3) pTrcKudzu yIDI DXS (kan) was used to inoculate 5 ml of LB pluskanamycin (50 μg/ml). The culture was incubated overnight at 25° C. withshaking. The following day the overnight culture was diluted to an OD₆₀₀of 0.05 in 25 ml of TM3 and 0.2% YE and 1% feedstock. The feedstock wascorn stover, bagasse, or softwood pulp. Glucose was used as a positivecontrol and no glucose was used as a negative control. Cultures wereincubated at 30° C. with shaking at 180 rpm. The culture was monitoredfor OD₆₀₀ and when it reached an OD₆₀₀ of ˜0.8, cultures were analyzedat 1 and 3 hours for isoprene production as described in Example 1.Cultures are not induced. All cultures containing added feedstockproduce isoprene equivalent to those of the glucose positive control.Experiments were done in duplicate and are shown in FIG. 46.

IV. Production of Isoprene from Invert Sugar in E. coli/pTrcKudzuIDIDXS

A single colony from a plate freshly transformed cells of BL21(λDE3)/pTrcKudzu yIDI DXS (kan) was used to inoculate 5 mL of LB andkanamycin (50 μg/ml). The culture was incubated overnight at 25° C. withshaking. The following day the overnight culture was diluted to an OD₆₀₀of 0.05 in 25 ml of TM3 and 0.2% YE and 1% feedstock. Feedstock wasglucose, inverted glucose or corn stover. The invert sugar feedstock(Danisco Invert Sugar) was prepared by enzymatically treating sucrosesyrup. AFEX corn stover was prepared as described below (Part V). Thecells were grown at 30° C. and the first sample was measured when thecultures reached an OD₆₀₀˜0.8-1.0 (0 hour). The cultures were analyzedfor growth as measured by OD₆₀₀ and for isoprene production as inExample 1 at 0, 1 and 3 hours. Results are shown in FIG. 47.

V. Preparation of Hydrolysate from AFEX Pretreated Corn Stover

AFEX pretreated corn stover was obtained from Michigan BiotechnologyInstitute. The pretreatment conditions were 60% moisture, 1:1 ammonialoading, and 90° C. for 30 minutes, then air dried. The moisture contentin the AFEX pretreated corn stover was 21.27%. The contents of glucanand xylan in the AFEX pretreated corn stover were 31.7% and 19.1% (drybasis), respectively. The saccharification process was as follows; 20 gof AFEX pretreated corn stover was added into a 500 ml flask with 5 mlof 1 M sodium citrate buffer pH 4.8, 2.25 ml of Accellerase 1000, 0.1 mlof Grindamyl H121 (Danisco xylanase product from Aspergillus niger forbread-making industry), and 72.65 ml of DI water. The flask was put inan orbital shaker and incubated at 50° C. for 96 hours. One sample wastaken from the shaker and analyzed using HPLC. The hydrolysate contained38.5 g/l of glucose, 21.8 g/l of xylose, and 10.3 g/l of oligomers ofglucose and/or xylose.

VI. The Effect of Yeast Extract on Isoprene Production in E. coli Grownin Fed-Batch Culture

Fermentation was performed at the 14-L scale as previously describedwith E. coli cells containing the pTrcKudzu yIDI DXS plasmid describedabove. Yeast extract (Bio Springer, Montreal, Quebec, Canada) was fed atan exponential rate. The total amount of yeast extract delivered to thefermentor was varied between 70-830 g during the 40 hour fermentation.Optical density of the fermentation broth was measured at a wavelengthof 550 nm. The final optical density within the fermentors wasproportional to the amount of yeast extract added (FIG. 48A). Theisoprene level in the off-gas from the fermentor was determined aspreviously described. The isoprene titer increased over the course ofthe fermentation (FIG. 48B). The amount of isoprene produced waslinearly proportional to the amount of fed yeast extract (FIG. 48C).

VII. Production of Isoprene in 500 L Fermentation of pTrcKudzu DXS yIDI

A 500 liter fermentation of E. coli cells with a kudzu isoprenesynthase, S. cerevisiae IDI, and E. coli DXS nucleic acids (E. coli BL21(XDE3) pTrc Kudzu dxs yidi) was used to produce isoprene. The levels ofisoprene varied from 50 to 300 μg/L over a time period of 15 hours. Onthe basis of the average isoprene concentrations, the average flowthrough the device and the extent of isoprene breakthrough, the amountof isoprene collected was calculated to be approximately 17 g.

VIII. Production of Isoprene in 500 L Fermentation of E. coli Grown inFed-Batch Culture

Medium Recipe (Per Liter Fermentation Medium):

K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferricammonium citrate 0.3 g, yeast extract 0.5 g, 1000× Modified Trace MetalSolution 1 ml. All of the components were added together and dissolvedin diH₂O. This solution was autoclaved. The pH was adjusted to 7.0 withammonium gas (NH₃) and q.s. to volume. Glucose 10 g, thiamine*HCl 0.1 g,and antibiotic were added after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

Citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g,CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg,NaMoO₄*2H₂O 100 mg. Each component is dissolved one at a time in DI H₂O,pH to 3.0 with HCl/NaOH, then q.s. to volume and filter sterilized with0.22 micron filter.

Fermentation was performed in a 500-L bioreactor with E. coli cellscontaining the pTrcKudzu yIDI DXS plasmid. This experiment was carriedout to monitor isoprene formation from glucose and yeast extract at thedesired fermentation pH 7.0 and temperature 30° C. An inoculum of E.coli strain taken from a frozen vial was prepared in soytone-yeastextract-glucose medium. After the inoculum grew to OD 0.15, measured at550 nm, 20 ml was used to inoculate a bioreactor containing 2.5-Lsoytone-yeast extract-glucose medium. The 2.5-L bioreactor was grown at30° C. to OD 1.0 and 2.0-L was transferred to the 500-L bioreactor.

Yeast extract (Bio Springer, Montreal, Quebec, Canada) and glucose werefed at exponential rates. The total amount of glucose and yeast extractdelivered to the bioreactor during the 50 hour fermentation was 181.2 kgand 17.6 kg, respectively. The optical density within the bioreactorover time is shown in FIG. 49A. The isoprene level in the off-gas fromthe bioreactor was determined as previously described. The isoprenetiter increased over the course of the fermentation (FIG. 49B). Thetotal amount of isoprene produced during the 50 hour fermentation was55.1 g and the time course of production is shown in FIG. 49C.

Example 8 Production of Isoprene in E. coli Expressing Kudzu IsopreneSynthase and Recombinant Mevalonic Acid Pathway Genes

I. Cloning the Lower MVA Pathway

The strategy for cloning the lower mevalonic pathway was as follows.Four genes of the mevalonic acid biosynthesis pathway; mevalonate kinase(MVK), phosphomevalonate kinase (PMK), diphosphomevalonte decarboxylase(MVD) and isopentenyl diphosphate isomerase genes were amplified by PCRfrom S. cerevisiae chromosomal DNA and cloned individually into the pCRBluntII TOPO plasmid (Invitrogen). In some cases, the idi gene wasamplified from E. coli chromosomal DNA. The primers were designed suchthat an E. coli consensus RBS (AGGAGGT (SEQ ID NO:80) or AAGGAGG (SEQ IDNO:81)) was inserted at the 5′ end, 8 bp upstream of the start codon anda PstI site was added at the 3′ end. The genes were then cloned one byone into the pTrcHis2B vector until the entire pathway was assembled.

Chromosomal DNA from S. cerevisiae S288C was obtained from ATCC (ATCC204508D). The MVK gene was amplified from the chromosome of S.cerevisiae using primers MVKF(5′-AGGAGGTAAAAAAACATGTCATTACCGTTCTTAACTTCTGC, SEQ ID NO:21) andMVK-Pst1-R (5′-ATGGCTGCAGGCCTATCGCAAATTAGCTTATGAAGTCCATGGTAAATTCGTG, SEQID NO:22) using PfuTurbo as per manufacturer's instructions. The correctsized PCR product (1370 bp) was identified by electrophoresis through a1.2% E-gel (Invitrogen) and cloned into pZeroBLUNT TOPO. The resultingplasmid was designated pMVK1. The plasmid pMVK1 was digested with SacIand Taq1 restriction endonucleases and the fragment was gel purified andligated into pTrcHis2B digested with SacI and BstBI. The resultingplasmid was named pTrcMVK1.

The second gene in the mevalonic acid biosynthesis pathway, PMK, wasamplified by PCR using primers: PstI-PMK1 R (5′-GAATTCGCCCTTCTGCAGCTACC,SEQ ID NO:23) and BsiHKA I-PMK1 F(5′-CGACTGGTGCACCCTTAAGGAGGAAAAAAACATGTCAG, SEQ ID NO:24). The PCRreaction was performed using Pfu Turbo polymerase (Stratagene) as permanufacturer's instructions. The correct sized product (1387 bp) wasdigested with PstI and BsiHKI and ligated into pTrcMVK1 digested withPstI. The resulting plasmid was named pTrcKK. The MVD and the idi geneswere cloned in the same manner. PCR was carried out using the primerpairs PstI-MVD 1 R (5′-GTGCTGGAATTCGCCCTTCTGCAGC, SEQ ID NO:25) andNsiI-MVD 1 F (5′-GTAGATGCATGCAGAATTCGCCCTTAAGGAGG, SEQ ID NO:26) toamplify the MVD gene and PstI-YIDI 1 R (5′-CCTTCTGCAGGACGCGTTGTTATAGC,SEQ ID NO:27) and NsiI-YIDI 1 F(5′-CATCAATGCATCGCCCTTAGGAGGTAAAAAAAAATGAC, SEQ ID NO:28) to amplify theyIDI gene. In some cases the IPP isomerase gene, idi from E. coli wasused. To amplify idi from E. coli chromosomal DNA, the following primerset was used: PstI-CIDI 1 R (5′-GTGTGATGGATATCTGCAGAATTCG, SEQ ID NO:29)and NsiI-CIDI 1 F (5′-CATCAATGCATCGCCCTTAGGAGGTAAAAAAACATG, SEQ IDNO:30). Template DNA was chromosomal DNA isolated by standard methodsfrom E. coli FM5 (WO 96/35796 and WO 2004/033646, which are each herebyincorporated by reference in their entireties, particularly with respectto isolation of nucleic acids). The final plasmids were named pKKDIy forthe construct encoding the yeast idi gene or pKKDIc for the constructencoding the E. coli idi gene. The plasmids were transformed into E.coli hosts BL21 for subsequent analysis. In some cases the isoprenesynthase from kudzu was cloned into pKKDIy yielding plasmid pKKDIyIS.

The lower MVA pathway was also cloned into pTrc containing a kanamycinantibiotic resistance marker. The plasmid pTrcKKDIy was digested withrestriction endonucleases ApaI and PstI, the 5930 bp fragment wasseparated on a 1.2% agarose E-gel and purified using the Qiagen GelPurification kit according to the manufacturer's instructions. Theplasmid pTrcKudzuKan, described in Example 7, was digested withrestriction endonucleases ApaI and PstI, and the 3338 bp fragmentcontaining the vector was purified from a 1.2% E-gel using the QiagenGel Purification kit. The 3338 bp vector fragment and the 5930 bp lowerMVA pathway fragment were ligated using the Roche Quick Ligation kit.The ligation mix was transformed into E. coli TOP10 cells andtransformants were grown at 37° C. overnight with selection on LAcontaining kanamycin (50 μg/ml). The transformants were verified byrestriction enzyme digestion and one was frozen as a stock. The plasmidwas designated pTrcKanKKDIy.

II. Cloning a Kudzu Isoprene Synthase Gene into pTrcKanKKDIy

The kudzu isoprene synthase gene was amplified by PCR from pTrcKudzu,described in Example 1, using primers MCM505′-GATCATGCATTCGCCCTTAGGAGGTAAAAAAACATGTGTGCGACCTCTTCTCAATTTACT (SEQ IDNO:31) and MCM53 5′-CGGTCGACGGATCCCTGCAGTTAGACATACATCAGCTG (SEQ IDNO:32). The resulting PCR fragment was cloned into pCR2.1 andtransformed into E. coli TOP10. This fragment contains the codingsequence for kudzu isoprene synthase and an upstream region containing aRBS from E. coli. Transformants were incubated overnight at 37° C. withselection on LA containing carbenicillin (50 μg/ml). The correctinsertion of the fragment was verified by sequencing and this strain wasdesignated MCM93.

The plasmid from strain MCM93 was digested with restrictionendonucleases NsiI and PstI to liberate a 1724 bp insert containing theRBS and kudzu isoprene synthase. The 1724 bp fragment was separated on a1.2% agarose E-gel and purified using the Qiagen Gel Purification kitaccording to the manufacturer's instructions. Plasmid pTrcKanKKDIy wasdigested with the restriction endonuclease PstI, treated with SAP for 30minutes at 37° C. and purified using the Qiagen PCR cleanup kit. Theplasmid and kudzu isoprene synthase encoding DNA fragment were ligatedusing the Roche Quick Ligation kit. The ligation mix was transformedinto E. coli TOP10 cells and transformants were grown overnight at 37°C. with selection on LA containing Kanamycin at 50 μg/ml. The correcttransformant was verified by restriction digestion and the plasmid wasdesignated pTrcKKDyIkISKan (FIGS. 24 and 25). This plasmid wastransformed into BL21(λDE3) cells (Invitrogen).

III. Isoprene Production from Mevalonate in E. coli Expressing theRecombinant Lower Mevalonate Pathway and Isoprene Synthase from Kudzu.

Strain BL21/pTrcKKDyIkISKan was cultured in MOPS medium (Neidhardt etal., (1974) J. Bacteriology 119:736-747) adjusted to pH 7.1 andsupplemented with 0.5% glucose and 0.5% mevalonic acid. A controlculture was also set up using identical conditions but without theaddition of 0.5% mevalonic acid. The culture was started from anovernight seed culture with a 1% inoculum and induced with 500 μM IPTGwhen the culture had reached an OD₆₀₀ of 0.3 to 0.5. The cultures weregrown at 30° C. with shaking at 250 rpm. The production of isoprene wasanalyzed 3 hours after induction by using the head space assay describedin Example 1. Maximum production of isoprene was 6.67×10⁴nmol/L_(broth)/OD₆₀₀/hr where L_(broth) is the volume of broth andincludes both the volume of the cell medium and the volume of the cells.The control culture not supplemented with mevalonic acid did not producemeasurable isoprene.

IV. Cloning the Upper MVA Pathway

The upper mevalonate biosynthetic pathway, comprising two genes encodingthree enzymatic activities, was cloned from Enterococcus faecalis. ThemvaE gene encodes a protein with the enzymatic activities of bothacetyl-CoA acetyltransferase and 3-hydroxy-3-methylglutaryl-CoA(HMG-CoA) reductase, the first and third proteins in the pathway, andthe mvaS gene encodes second enzyme in the pathway, HMG-CoA synthase.The mvaE gene was amplified from E. faecalis genomic DNA (ATCC700802D-5) with an E. coli ribosome binding site and a spacer in frontusing the following primers:

CF 07-60 (+) Start of mvaE w/RBS + ATG start codon SacI (SEQ ID NO: 34)5′-GAGACATGAGCTCAGGAGGTAAAAAAACATGAAAACAGTAGTTATT ATTGCF 07-62 (−) Fuse mvaE to mvaS with RBS in between (SEQ ID NO: 35)5′-TTTATCAATCCCAATTGTCATGTTTTTTTACCTCCTTTATTGTTTT CTTAAATC

The mvaS gene was amplified from E. faecalis genomic DNA (ATCC700802D-5) with a RBS and spacer from E. coli in front using thefollowing primers:

CF 07-61 (+) Fuse mvaE to mvaS with RBS in between (SEQ ID NO: 36)5′-GATTTAAGAAAACAATAAAGGAGGTAAAAAAACATGACAATTGGGA TTGATAAACF 07-102 (−) End of mvaS gene BglII (SEQ ID NO: 37)5′-GACATGACATAGATCTTTAGTTTCGATAAGAACGAACGGT

The PCR fragments were fused together with PCR using the followingprimers:

CF 07-60 (+) Start of mvaE w/RBS + ATG start codon SacI (SEQ ID NO: 34)5′-GAGACATGAGCTCAGGAGGTAAAAAAACATGAAAACAGTAGTTATT ATTGCF 07-102 (−) End of mvaS gene BglII (SEQ ID NO: 37)5′-GACATGACATAGATCTTTAGTTTCGATAAGAACGAACGGT

The fusion PCR fragment was purified using a Qiagen kit and digestedwith the restriction enzymes SacI and BglII. This digested DNA fragmentwas gel purified using a Qiagen kit and ligated into the commerciallyavailable vector pTrcHis2A, which had been digested with SacI and BglIIand gel purified.

The ligation mix was transformed into E. coli Top 10 cells and colonieswere selected on LA and 50 μg/ml carbenicillin plates. A total of sixcolonies were chosen and grown overnight in LB and 50 μg/mlcarbenicillin and plasmids were isolated using a Qiagen kit. Theplasmids were digested with SacI and BglII to check for inserts and onecorrect plasmid was sequenced with the following primers:

CF 07-58 (+) Start of mvaE gene (SEQ ID NO: 38)5′-ATGAAAACAGTAGTTATTATTGATGC CF 07-59 (−) End of mvaE gene(SEQ ID NO: 39) 5′-ATGTTATTGTTTTCTTAAATCATTTAAAATAGCCF 07-82 (+) Start of mvaS gene (SEQ ID NO: 40)5′-ATGACAATTGGGATTGATAAAATTAG CF 07-83 (−) End of mvaS gene(SEQ ID NO: 41) 5′-TTAGTTTCGATAAGAACGAACGGTCF 07-86 (+) Sequence in mvaE (SEQ ID NO: 42) 5′-GAAATAGCCCCATTAGAAGTATCCF 07-87 (+) Sequence in mvaE (SEQ ID NO: 43)5′-TTGCCAATCATATGATTGAAAATC CF 07-88 (+) Sequence in mvaE(SEQ ID NO: 44) 5′-GCTATGCTTCATTAGATCCTTATCG CF 07-89 (+) Sequence mvaS(SEQ ID NO: 45) 5′-GAAACCTACATCCAATCTTTTGCCC

The plasmid called pTrcHis2AUpperPathway#1 was correct by sequencing andwas transformed into the commercially available E. coli strain BL21.Selection was done on LA and 50 μg/ml carbenicillin. Two transformantswere chosen and grown in LB and 50 μg/ml carbenicillin until theyreached an OD₆₀₀ of 1.5. Both strains were frozen in a vial at −80° C.in the presence of glycerol. Strains were designated CF 449 forpTrcHis2AUpperPathway#1 in BL21, isolate #1 and CF 450 forpTrcHis2AUpperPathway#1 in BL21, isolate #2. Both clones were found tobehave identically when analyzed.

V. Cloning of UpperMVA Pathway into pCL1920

The plasmid pTrcHis2AUpperPathway was digested with the restrictionendonuclease SspI to release a fragment containing pTrc-mvaE-mvaS-(Histag)-terminator. In this fragment, the his-tag was not translated. Thisblunt ended 4.5 kbp fragment was purified from a 1.2% E-gel using theQiagen Gel Purification kit. A dephosphorylated, blunt ended 4.2 kbpfragment from pCL1920 was prepared by digesting the vector with therestriction endonuclease PvuII, treating with SAP and gel purifying froma 1.2% E-gel using the Qiagen Gel Purification kit. The two fragmentswere ligated using the Roche Quick Ligation Kit and transformed intoTOP10 chemically competent cells. Transformants were selected on LAcontaining spectinomycin (50 μg/ml). A correct colony was identified byscreening for the presence of the insert by PCR. The plasmid wasdesignated pCL PtrcUpperPathway (FIGS. 26 and 27).

VI. Strains Expressing the Combined Upper and Lower Mevalonic AcidPathways

To obtain a strain with a complete mevalonic acid pathway plus kudzuisoprene synthase, plasmids pTrcKKDyIkISkan and pCLpTrcUpperPathway wereboth transformed into BL21(λDE3) competent cells (Invitrogen) andtransformants were selected on LA containing kanamycin (50 μg/ml) andSpectinomycin (50 μg/ml). The transformants were checked by plasmid prepto ensure that both plasmids were retained in the host. The strain wasdesignated MCM127.

VII. Production of Mevalonic Acid from Glucose in E. coli/pUpperpathway

Single colonies of the BL21/pTrcHis2A-mvaE/mvaS or FM5/ppTrcHis2A-mvaE/mvaS are inoculated into LB and carbenicillin (100 μg/ml)and are grown overnight at 37° C. with shaking at 200 rpm. Thesecultures were diluted into 50 ml medium in 250 ml baffled flasks to anOD₆₀₀ of 0.1. The medium was TM3, 1 or 2% glucose, carbenicillin (100ug/ml) or TM3, 1% glucose, hydrolyzed soy oil, and carbenicillin (100ug/ml) or TM3 and biomass (prepared bagasse, corn stover orswitchgrass). Cultures were grown at 30° C. with shaking at 200 rpm forapproximately 2-3 hours until an OD₆₀₀ of 0.4 was reached. At this pointthe expression from the mvaE mvaS construct was induced by the additionof IPTG (400 μM). Cultures were incubated for a further 20 or 40 hourswith samples taken at 2 hour intervals to 6 hour post induction and thenat 24, 36 and 48 hours as needed. Sampling was done by removing 1 ml ofculture, measuring the OD₆₀₀, pelleting the cells in a microfuge,removing the supernatant and analyzing it for mevalonic acid.

A 14 liter fermentation of E. coli cells with nucleic acids encodingEnterococcus faecalis AA-CoA thiolase, HMG-CoA synthase, and HMG-CoAreductase polypeptides produced 22 grams of mevalonic acid with TM3medium and 2% glucose as the cell medium. A shake flask of these cellsproduced 2-4 grams of mevalonic acid per liter with LB medium and 1%glucose as the cell culture medium. The production of mevalonic acid inthese strains indicated that the MVA pathway was functional in E. coli.

VIII. Production of Isoprene from E. coli BL21 Containing the Upper andLower MVA Pathway Plus Kudzu Isoprene Synthase.

The following strains were created by transforming in variouscombinations of plasmids containing the upper and lower MVA pathway andthe kudzu isoprene synthase gene as described above and the plasmidscontaining the idi, dxs, and dxr and isoprene synthase genes describedin Example 7. The host cells used were chemically competent BL21(λDE3)and the transformations were done by standard methods. Transformantswere selected on L agar containing kanamycin (50 μg/ml) or kanamycinplus spectinomycin (both at a concentration of 50 μg/ml). Plates weregrown at 37° C. The resulting strains were designated as follows:

Grown on Kanamycin Plus Spectinomycin (50 μg/ml Each)

-   MCM127-pCL Upper MVA and pTrcKKDyIkIS (kan) in BL21(λDE3)-   MCM131-pCL1920 and pTrcKKDyIkIS (kan) in BL21(λDE3)-   MCM125-pCL Upper MVA and pTrcHis2B (kan) in BL21(λDE3)    Grown on Kanamycin (50 μg/ml)-   MCM64-pTrcKudzu yIDI DXS (kan) in BL21(λDE3)-   MCM50-pTrcKudzu (kan) in BL21(λDE3)-   MCM123-pTrcKudzu yIDI DXS DXR (kan) in BL21(λDE3)

The above strains were streaked from freezer stocks to LA andappropriate antibiotic and grown overnight at 37° C. A single colonyfrom each plate was used to inoculate shake flasks (25 ml LB and theappropriate antibiotic). The flasks were incubated at 22° C. overnightwith shaking at 200 rpm. The next morning the flasks were transferred toa 37° C. incubator and grown for a further 4.5 hours with shaking at 200rpm. The 25 ml cultures were centrifuged to pellet the cells and thecells were resuspended in 5 ml LB and the appropriate antibiotic. Thecultures were then diluted into 25 ml LB, % glucose, and the appropriateantibiotic to an OD₆₀₀ of 0.1. Two flasks for each strain were set up,one set for induction with IPTG (800 μM) the second set was not induced.The cultures were incubated at 37° C. with shaking at 250 rpm. One setof the cultures were induced after 1.50 hours (immediately followingsampling time point 1). At each sampling time point, the OD₆₀₀ wasmeasured and the amount of isoprene determined as described inExample 1. Results are presented in Table 3. The amount of isoprene madeis presented as the amount at the peak production for the particularstrain.

TABLE 3 Production of isoprene in E. coli strains Strain Isoprene(μg/L_(broth)/hr/OD MCM50 23.8 MCM64 289 MCM125 ND MCM131 Trace MCM127874 ND: not detected Trace: peak present but not integrable.IX. Analysis of Mevalonic Acid

Mevalonolactone (1.0 g, 7.7 mmol) (CAS #503-48-0) was supplied fromSigma-Aldrich (WI, USA) as a syrup that was dissolved in water (7.7 mL)and was treated with potassium hydroxide (7.7 mmol) in order to generatethe potassium salt of mevalonic acid. The conversion to mevalonic acidwas confirmed by ¹H NMR analysis. Samples for HPLC analysis wereprepared by centrifugation at 14,000 rpm for 5 minutes to remove cells,followed by the addition of a 300 μl aliquot of supernatant to 900 μl ofH₂O. Perchloric acid (36 μl of a 70% solution) was then added followedby mixing and cooling on ice for 5 minutes. The samples were thencentrifuged again (14,000 rpm for 5 min) and the supernatant transferredto HPLC. Mevalonic acid standards (20, 10, 5, 1 and 0.5 g/L) wereprepared in the same fashion. Analysis of mevalonic acid (20 uLinjection volume) was performed by HPLC using a BioRad Aminex 87-H+column (300 mm by 7.0 mm) eluted with 5 mM sulfuric acid at 0.6 mL/minwith refractive index (RI) detection. Under these conditions mevalonicacid eluted as the lactone form at 18.5 minutes.

Example 9 Construction of the Upper and Lower MVA Pathway forIntegration into Bacillus subtilis

I. Construction of the Upper MVA Pathway in Bacillus subtilis

The upper pathway from Enterococcus faecalis is integrated into B.subtilis under control of the aprE promoter. The upper pathway consistsof two genes; mvaE, which encodes for AACT and HMGR, and mvaS, whichencodes for HMGS. The two genes are fused together with a stop codon inbetween, an RBS site in front of mvaS, and are under the control of theaprE promoter. A terminator is situated after the mvaE gene. Thechloramphenicol resistance marker is cloned after the mvaE gene and theconstruct is integrated at the aprE locus by double cross over usingflanking regions of homology.

Four DNA fragments are amplified by PCR such that they contain overhangsthat will allow them to be fused together by a PCR reaction. PCRamplifications are carried out using Herculase polymerase according tomanufacturer's instructions.

1: PaprE

CF 07-134 (+) Start of aprE promotoer PstI (SEQ ID NO: 82)5′-GACATCTGCAGCTCCATTTTCTTCTGC CF 07-94 (−) Fuse PaprE to mvaE(SEQ ID NO: 83) 5′-CAATAATAACTACTGTTTTCACTCTTTACCCTCTCCTTTTAATemplate: Bacillus subtilis chromosomal DNA 2: mvaE

CF 07-93 (+) fuse mvaE to the aprE promoter (GTG start codon)(SEQ ID NO: 84) 5′-TTAAAAGGAGAGGGTAAAGAGTGAAAACAGTAGTTATTATTGCF 07-62 (−) Fuse mvaE to mvaS with RBS in between (SEQ ID NO: 35)5′-TTTATCAATCCCAATTGTCATGTTTTTTTACCTCCTTTATTGTTTT CTTAAATCTemplate: Enterococcus faecalis chromosomal DNA (from ATCC)3. mvaS

CF 07-61 (+) Fuse mvaE to mvaS with RBS in between (SEQ ID NO: 36)5′-GATTTAAGAAAACAATAAAGGAGGTAAAAAAACATGACAATTGGGA TTGATAAACF 07-124 (−) Fuse the end of mvaS to the terminator (SEQ ID NO: 85)5′-CGGGGCCAAGGCCGGTTTTTTTTAGTTTCGATAAGAACGAACGGTTemplate: Enterococcus faecalis chromosomal DNA4. B. amyliquefaciens alkaline serine protease terminator

CF 07-123 (+) Fuse the end of mvaS to the terminator (SEQ ID NO: 86)5′-ACCGTTCGTTCTTATCGAAACTAAAAAAAACCGGCCTTGGCCCCGCF 07-46 (−) End if B. amyliquefaciens terminator BamHI (SEQ ID NO: 63)5′-GACATGACGGATCCGATTACGAATGCCGTCTCTemplate: Bacillus amyliquefaciens chromosomal DNAPCR Fusion Reactions5. Fuse mvaE to mvaS

CF 07-93 (+) fuse mvaE to the aprE promotoer (GTG start codon)(SEQ ID NO: 84) 5′-TTAAAAGGAGAGGGTAAAGAGTGAAAACAGTAGTTATTATTGCF 07-124 (−) Fuse the end of mvaS to the terminator (SEQ ID NO: 85)5′-CGGGGCCAAGGCCGGTTTTTTTTAGTTTCGATAAGAACGAACGGTTemplate: #2 and 3 from above6. Fuse mvaE-mvaS to aprE promoter

CF 07-134 (+) Start of aprE promoter PstI (SEQ ID NO: 82)5′-GACATCTGCAGCTCCATTTTCTTCTGC CF 07-124 (−) Fuse the end of mvaS to theterminator (SEQ ID NO: 85)5′-CGGGGCCAAGGCCGGTTTTTTTTAGTTTCGATAAGAACGAACGGTTemplate #1 and #4 from above7. Fuse PaprE-mvaE-mvaS to terminator

CF 07-134 (+) Start of aprE promoter PstI (SEQ ID NO: 82)5′-GACATCTGCAGCTCCATTTTCTTCTGCCF 07-46 (−) End of B. amyliquefaciens terminator BamHI (SEQ ID NO: 63)5′-GACATGACGGATCCGATTACGAATGCCGTCTCTemplate: #4 and #6

The product is digested with restriction endonucleases PstI/BamHI andligated to pJM102 (Perego, M. 1993. Integrational vectors for geneticmanipulation in Bacillus subtilis, p. 615-624. In A. L. Sonenshein, J.A. Hoch, and R. Losick (ed.), Bacillus subtilis and other gram-positivebacteria: biochemistry, physiology, and molecular genetics. AmericanSociety for Microbiology, Washington, D.C.) which is digested withPstI/BamHI. The ligation is transformed into E. coli TOP 10 chemicallycompetent cells and transformants are selected on LA containingcarbenicillin (50 μg/ml). The correct plasmid is identified bysequencing and is designated pJMUpperpathway2 (FIGS. 50 and 51).Purified plasmid DNA is transformed into Bacillus subtilis aprEnprEPxyl-comK and transformants are selected on L agar containingchloramphenicol (5 μg/ml). A correct colony is selected and is platedsequentially on L agar containing chloramphenicol 10, 15 and 25 μg/ml toamplify the number of copies of the cassette containing the upperpathway.

The resulting strain is tested for mevalonic acid production by growingin LB containing 1% glucose and 1%. Cultures are analyzed by GC for theproduction of mevalonic acid.

This strain is used subsequently as a host for the integration of thelower mevalonic acid pathway.

The following primers are used to sequence the various constructs above.

Sequencing Primers:

CF 07-134 (+) Start of aprE promoter PstI (SEQ ID NO: 82)5′-GACATCTGCAGCTCCATTTTCTTCTGC CF 07-58 (+) Start of mvaE gene(SEQ ID NO: 38) 5′-ATGAAAACAGTAGTTATTATTGATGCCF 07-59 (−) End of mvaE gene (SEQ ID NO: 39)5′-ATGTTATTGTTTTCTTAAATCATTTAAAATAGC CF 07-82 (+) Start of mvaS gene(SEQ ID NO: 40) 5′-ATGACAATTGGGATTGATAAAATTAGCF 07-83 (−) End of mvaS gene (SEQ ID NO: 41)5′-TTAGTTTCGATAAGAACGAACGGT CF 07-86 (+) Sequence in mvaE(SEQ ID NO: 42) 5′-GAAATAGCCCCATTAGAAGTATC CF 07-87 (+) Sequence in mvaE(SEQ ID NO: 43) 5′-TTGCCAATCATATGATTGAAAATCCF 07-88 (+) Sequence in mvaE (SEQ ID NO: 44)5′-GCTATGCTTCATTAGATCCTTATCG CF 07-89 (+) Sequence mvaS (SEQ ID NO: 45)5′-GAAACCTACATCCAATCTTTTGCCC

Transformants are selected on LA containing chloramphenicol at aconcentration of 5 μg/ml. One colony is confirmed to have the correctintegration by sequencing and is plated on LA containing increasingconcentrations of chloramphenicol over several days, to a final level of25 μg/ml. This results in amplification of the cassette containing thegenes of interest. The resulting strain is designated CF 455:pJMupperpathway#1 X Bacillus subtilis aprEnprE Pxyl comK (amplified togrow on LA containing chloramphenicol 25 μg/ml).

II. Construction of the Lower MVA Pathway in Bacillus subtilis

The lower MVA pathway, consisting of the genes mvk1, pmk, mpd and idiare combined in a cassette consisting of flanking DNA regions from thenprE region of the B. subtilis chromosome (site of integration), theaprE promoter, and the spectinomycin resistance marker (see FIGS. 28 and29). This cassette is synthesized by DNA2.0 and is integrated into thechromosome of B. subtilis containing the upper MVA pathway integrated atthe aprE locus. The kudzu isoprene synthase gene is expressed from thereplicating plasmid described in Example 4 and is transformed into thestrain with both upper and lower pathways integrated.

Example 10 Production of Isoprene in E. coli Expressing M. mazeiMevalonate Kinase and P. alba Isoprene Synthase

I. Construction of Vectors and Strains Encoding M. mazei MevalonateKinase (MVK) and P. alba Isoprene Synthase

(i) Construction of Strain EWL201 (BL21, Cm-GI1.2-KKDyI)

E. coli BL21 (Novagen brand, EMD Biosciences, Inc.) was a recipientstrain, transduced with MCM331 P1 lysate (lysate prepared according tothe method described in Ausubel, et al., Current Protocols in MolecularBiology. John Wiley and Sons, Inc.). Transductants were selected for byspreading cells onto L Agar and 20 μg/μl chloramphenicol. The plateswere incubated overnight at 30° C. Analysis of transductants showed nocolonies on control plates (water+cells control plate for reversion andwater and P1 lysate control plate for lysate contamination.

Four transductants were picked and used to inoculate 5 mL L Broth and 20μg/μl chloramphenicol. The cultures were grown overnight at 30° C. withshaking at 200 rpm. To make genomic DNA preps of each transductant forPCR analysis, 1.5 mL of overnight cell culture were centrifuged. Thecell pellet was resuspended with 400 μl Resuspension Buffer (20 mM Tris,1 mM EDTA, 50 mM NaCl, pH 7.5) and 4 μl RNase, DNase-free (Roche) wasadded. The tubes were incubated at 37° C. for 30 minutes followed by theaddition of 4 μl 10% SDS and 4 μl of 10 mg/ml Proteinase K stocksolution (Sigma-Aldrich). The tubes were incubated at 37° C. for 1 hour.The cell lysate was transferred into 2 ml Phase Lock Light Gel tubes(Eppendorf) and 200 μl each of saturated phenol pH7.9 (Ambion Inc.) andchloroform were added. The tubes were mixed well and microcentrifugedfor 5 minutes. A second extraction was done with 400 μl chloroform andthe aqueous layer was transferred to a new eppendorf tube. The genomicDNA was precipitated by the addition of 1 ml of 100% ethanol andcentrifugation for 5 minutes. The genomic DNA pellet was washed with 1ml 70% ethanol. The ethanol was removed and the genomic DNA pellet wasallowed to air dry briefly. The genomic DNA pellet was resuspended with200 μl TE.

Using Pfu Ultra II DNA polymerase (Stratagene) and 200 ng/μl of genomicDNA as template, 2 different sets of PCR reaction tubes were preparedaccording to manufacturer's protocol. For set 1, primers MCM130 and GBCm-Rev (Table 4) were used to ensure transductants were successfullyintegrated into the attTn7 locus. PCR parameters for set 1 were 95° C.for 2 minutes (first cycle only), 95° C. for 25 seconds, 55° C. for 25seconds, 72° C. for 25 seconds (repeat steps 2-4 for 28 cycles), 72° C.for 1 minute. For set 2, primers MVD For and MVD Rev (Table 4) were usedto ensure that the gi1.2-KKDyI operon integrated properly. PCRparameters for set 2 were 95° C. for 2 minutes (first cycle only), 95°C. for 25 seconds, 55° C. for 25 seconds, 72° C. for 10 seconds (repeatsteps 2-4 for 28 cycles), 72° C. for 1 minute. Analysis of PCR ampliconson a 1.2% E-gel (Invitrogen Corp.) showed that all 4 transductant cloneswere correct (picked one and designated as strain EWL201).

ii) Construction of Strain EWL204 (BL21, Loopout-GI1.2-KKDyI)

The chloramphenicol marker was looped out of strain EWL201 using plasmidpCP20 as described by Datsenko and Wanner (2000) (Datsenko et al., ProcNatl. Acad. Sci USA 97:6640-6645, 2000). One-step inactivation ofchromosomal genes in Escherichia coli K-12 using PCR products. (Datsenkoet al., PNAS, 97: 6640-6645, 2000). EWL201 cells were grown in L Brothto midlog phase and then washed three times in ice-cold, sterile water.An aliquot of 50 μl of cell suspension was mixed with 1 μl of pCP20 andthe cell suspension mixture was electroporated in a 2 mm cuvette(Invitrogen Corp.) at 2.5 Volts and 25 uFd using a Gene PulserElectroporator (Bio-Rad Inc.). 1 ml of LB was immediately added to thecells, then transferred to a 14 ml polypropylene tube (Sarstedt) with ametal cap. Cells were allowed to recover by growing for 1 hour at 30° C.Transformants were selected on L Agar and 20 μg/μl chloramphenicol and50 μg/μl carbenicillin and incubated at 30° C. overnight. The next day,a single clone was grown in 10 ml L Broth and 50 μg/μl carbenicillin at30° C. until early log phase. The temperature of the growing culture wasthen shifted to 42° C. for 2 hours. Serial dilutions were made, thecells were then spread onto LA plates (no antibiotic selection), andincubated overnight at 30° C. The next day, 20 colonies were picked andpatched onto L Agar (no antibiotics) and LA and 20 μg/μl chloramphenicolplates. Plates were then incubated overnight at 30° C. Cells able togrow on LA plates, but not LA and 20 μg/μl chloramphenicol plates, weredeemed to have the chloramphenicol marker looped out (picked one anddesignated as strain EWL204).

iii) Construction of Plasmid pEWL230 (pTrc P. alba)

Generation of a synthetic gene encoding Populus alba isoprene synthase(P. alba HGS) was outsourced to DNA2.0 Inc. (Menlo Park, Calif.) basedon their codon optimization method for E. coli expression. The syntheticgene was custom cloned into plasmid pET24a (Novagen brand, EMDBiosciences, Inc.) and delivered lyophilized (FIGS. 54, 55A and 55B).

A PCR reaction was performed to amplify the P. alba isoprene synthase(P. alba HGS) gene using pET24 P. alba HGS as the template, primersMCM182 and MCM192, and Herculase II Fusion DNA polymerase (Stratagene)according to manufacturer's protocol. PCR conditions were as follows:95° C. for 2 minutes (first cycle only), 95° C. for 25 seconds, 55° C.for 20 seconds, 72° C. for 1 minute, repeat for 25 cycles, with finalextension at 72° C. for 3 minutes. The P. alba isoprene synthase PCRproduct was purified using QIAquick PCR Purification Kit (Qiagen Inc.).

P. alba isoprene synthase PCR product was then digested in a 20 μlreaction containing 1 μl BspHI endonuclease (New England Biolabs) with 2μl 10×NEB Buffer 4. The reaction was incubated for 2 hours at 37° C. Thedigested PCR fragment was then purified using the QIAquick PCRPurification Kit. A secondary restriction digest was performed in a 20μl reaction containing 1 μl PstI endonuclease (Roche) with 2 μl 10×Buffer H. The reaction was incubated for 2 hours at 37° C. The digestedPCR fragment was then purified using the QIAquick PCR Purification Kit.Plasmid pTrcHis2B (Invitrogen Corp.) was digested in a 20 μl reactioncontaining 1 μl NcoI endonuclease (Roche), 1 μl PstI endonuclease, and 2μl 10× Buffer H. The reaction was incubated for 2 hours at 37° C. Thedigested pTrcHis2B vector was gel purified using a 1.2% E-gel(Invitrogen Corp.) and extracted using the QIAquick Gel Extraction Kit(Qiagen) (FIG. 56). Using the compatible cohesive ends of BspHI and NcoIsites, a 20 μl ligation reaction was prepared containing 5 μl P. albaisoprene synthase insert, 2 μl pTrc vector, 1 μl T4 DNA ligase (NewEngland Biolabs), 2 μl 10× ligase buffer, and 10 μl ddH₂O. The ligationmixture was incubated at room temperature for 40 minutes. The ligationmixture was desalted by floating a 0.025 μm nitrocellulose membranefilter (Millipore) in a petri dish of ddH₂O and applying the ligationmixture gently on top of the nitrocellulose membrane filter for 30minutes at room temperature. MCM446 cells (See section II) were grown inLB to midlog phase and then washed three times in ice-cold, sterilewater. An aliquot of 50 μl of cell suspension was mixed with 5 μl ofdesalted pTrc P. alba HGS ligation mix. The cell suspension mixture waselectroporated in a 2 mm cuvette at 2.5 Volts and 25 uFd using a GenePulser Electroporator. 1 ml of LB is immediately added to the cells,then transferred to a 14 ml polypropylene tube (Sarstedt) with a metalcap. Cells were allowed to recover by growing for 2 hour at 30° C.Transformants were selected on L Agar and 50 μg/μl carbenicillin and 10mM mevalonic acid and incubated at 30° C. The next day, 6 transformantswere picked and grown in 5 ml L Broth and 50 μg/μl carbenicillin tubesovernight at 30° C. Plasmid preps were performed on the overnightcultures using QIAquick Spin Miniprep Kit (Qiagen). Due to the use ofBL21 cells for propagating plasmids, a modification of washing the spincolumns with PB Buffer 5× and PE Buffer 3× was incorporated to thestandard manufacturer's protocol for achieving high quality plasmid DNA.Plasmids were digested with PstI in a 20 μl reaction to ensure thecorrect sized linear fragment. All 6 plasmids were the correct size andshipped to Quintara Biosciences (Berkeley, Calif.) for sequencing withprimers MCM65, MCM66, EL1000 (Table 4). DNA sequencing results showedall 6 plasmids were correct. Picked one and designated plasmid as EWL230(FIGS. 57, 58A and 58B).

iv) Construction of Plasmid pEWL244 (pTrc P. alba-mMVK)

A PCR reaction was performed to amplify the Methanosarcina mazei (M.mazei) MVK gene using MCM376 as the template (see section v), primersMCM165 and MCM177 (see Table 4), and Pfu Ultra II Fusion DNA polymerase(Stratagene) according to manufacturer's protocol. PCR conditions wereas follows: 95° C. for 2 minutes (first cycle only), 95° C. for 25seconds, 55° C. for 25 seconds, 72° C. for 18 seconds, repeat for 28cycles, with final extension at 72° C. for 1 minute. The M. mazei MVKPCR product was purified using QIAquick PCR Purification Kit (QiagenInc.)

The M. mazei MVK PCR product was then digested in a 40 μl reactioncontaining 8 μl PCR product, 2 μl PmeI endonuclease (New EnglandBiolabs), 4 μl 10×NEB Buffer 4, 4 μl 10×NEB BSA, and 22 μl of ddH₂O. Thereaction was incubated for 3 hours at 37° C. The digested PCR fragmentwas then purified using the QIAquick PCR Purification Kit. A secondaryrestriction digest was performed in a 47 μl reaction containing 2 μlNsiI endonuclease (Roche), 4.7 μl 10× Buffer H, and 40 μl of PmeIdigested M. mazei MVK fragment. The reaction was incubated for 3 hoursat 37° C. The digested PCR fragment was then gel purified using a 1.2%E-gel and extracted using the QIAquick Gel Extraction Kit. PlasmidEWL230 was digested in a 40 μl reaction containing 10 μl plasmid, 2 μlPmeI endonuclease, 4 μl 10×NEB Buffer 4, 4 μl 10×NEB BSA, and 20 μl ofddH₂O. The reaction was incubated for 3 hours at 37° C. The digested PCRfragment was then purified using the QIAquick PCR Purification Kit. Asecondary restriction digest was performed in a 47 μl reactioncontaining 2 μl PstI endonuclease, 4.7 μl 10× Buffer H, and 40 μl ofPmeI digested EWL230 linear fragment. The reaction was incubated for 3hours at 37° C. The digested PCR fragment was then gel purified using a1.2% E-gel and extracted using the QIAquick Gel Extraction Kit (FIG.59). Using the compatible cohesive ends of NsiI and PstI sites, a 20 μlligation reaction was prepared containing 8 μl M. mazei MVK insert, 3 μlEWL230 plasmid, 1 μl T4 DNA ligase, 2 μl 10× ligase buffer, and 6 μlddH₂O. The ligation mixture was incubated at overnight at 16° C. Thenext day, the ligation mixture was desalted by floating a 0.025 μmnitrocellulose membrane filter in a petri dish of ddH₂O and applying theligation mixture gently on top of the nitrocellulose membrane filter for30 minutes at room temperature. MCM446 cells were grown in LB to midlogphase and then washed three times in ice-cold, sterile water. An aliquotof 50 μl of cell suspension was mixed with 5 μl of desalted pTrc P.alba-mMVK ligation mix. The cell suspension mixture was electroporatedin a 2 mm cuvette at 2.5 Volts and 25 uFd using a Gene PulserElectroporator. 1 ml of LB is immediately added to the cells, then thecells are transferred to a 14 ml polypropylene tube with a metal cap.Cells were allowed to recover by growing for 2 hour at 30° C.Transformants were selected on LA and 50 μg/μl carbenicillin and 5 mMmevalonic acid plates and incubated at 30° C. The next day, 6transformants were picked and grown in 5 ml LB and 50 μg/μlcarbenicillin tubes overnight at 30° C. Plasmid preps were performed onthe overnight cultures using QIAquick Spin Miniprep Kit. Due to the useof BL21 cells for propagating plasmids, a modification of washing thespin columns with PB Buffer 5× and PE Buffer 3× was incorporated to thestandard manufacturer's protocol for achieving high quality plasmid DNA.Plasmids were digested with PstI in a 20 μl reaction to ensure thecorrect sized linear fragment. Three of the 6 plasmids were the correctsize and shipped to Quintara Biosciences for sequencing with primersMCM65, MCM66, EL1000, EL1003, and EL1006 (Table 4). DNA sequencingresults showed all 3 plasmids were correct. Picked one and designatedplasmid as EWL244 (FIGS. 60 and 61A-B).

v) Construction of Plasmid MCM376-MVK from M. mazei Archaeal Lower inpET200D.

The MVK ORF from the M. mazei archaeal Lower Pathway operon (FIGS.73A-C) was PCR amplified using primers MCM161 and MCM162 (Table 4) usingthe Invitrogen Platinum HiFi PCR mix. 45 uL of PCR mix was combined with1 uL template, 1 uL of each primer at 10 uM, and 2 uL water. Thereaction was cycled as follows: 94° C. for 2:00 minutes; 30 cycles of94° C. for 0:30 minutes, 55° C. for 0:30 minutes and 68° C. for 1:15minutes; and then 72° C. for 7:00 minutes, and 4° C. until cool. 3 uL ofthis PCR reaction was ligated to Invitrogen pET200D plasmid according tothe manufacturer's protocol. 3 uL of this ligation was introduced intoInvitrogen TOP10 cells, and transformants were selected on LA/kan50. Aplasmid from a transformant was isolated and the insert sequenced,resulting in MCM376 (FIGS. 74A-C).

vi) Construction of Strain EWL251 (BL21(DE3), Cm-GI1.2-KKDyI, pTrcP.alba-mMVK)

MCM331 cells (which contain chromosomal construct gi1.2KKDyI encoding S.cerevisiae mevalonate kinase, mevalonate phosphate kinase, mevalonatepyrophosphate decarboxylase, and IPP isomerase) were grown in LB tomidlog phase and then washed three times in ice-cold, sterile water.Mixed 50 μl of cell suspension with 1 μl of plasmid EWL244. The cellsuspension mixture was electroporated in a 2 mm cuvette at 2.5 Volts and25 uFd using a Gene Pulser Electroporator. 1 ml of LB is immediatelyadded to the cells, then the cells were transferred to a 14 mlpolypropylene tube with a metal cap. Cells were allowed to recover bygrowing for 2 hours at 30° C. Transformants were selected on LA and 50μg/μl carbenicillin and 5 mM mevalonic acid plates and incubated at 37°C. One colony was selected and designated as strain EWL251.

vii) Construction of Strain EWL256 (BL21(DE3), Cm-GI1.2-KKDyI, pTrc P.alba-mMVK, pCL Upper MVA)

EWL251 cells were grown in LB to midlog phase and then washed threetimes in ice-cold, sterile water. Mixed 50 μl of cell suspension with 1μl of plasmid MCM82 (which is pCL PtrcUpperPathway encoding E. faecalismvaE and mvaS). The cell suspension mixture was electroporated in a 2 mmcuvette at 2.5 Volts and 25 uFd using a Gene Pulser Electroporator. 1 mlof LB was immediately added to the cells, then transferred to a 14 mlpolypropylene tube with a metal cap. Cells were allowed to recover bygrowing for 2 hour at 30° C. Transformants were selected on LA and 50μg/μl carbenicillin and 50 μg/μl spectinomycin plates and incubated at37° C. Picked one colony and designated as strain EWL256.

TABLE 4 Primer Sequences Primer name Primer sequence MCM130ACCAATTGCACCCGGCAGA (SEQ ID NO: 94) GB Cm GCTAAAGCGCATGCTCCAGAC Rev(SEQ ID NO: 95) MVD GACTGGCCTCAGATGAAAGC For (SEQ ID NO: 96) MVDCAAACATGTGGCATGGAAAG Rev (SEQ ID NO: 97) MCM182GGGCCCGTTTAAACTTTAACTAGACTCTGCAGTTAGCGTTCAAA CGGCAGAA (SEQ ID NO: 98)MCM192 CGCATGCATGTCATGAGATGTAGCGTGTCCACCGAAAA (SEQ ID NO: 99 MCM65ACAATTTCACACAGGAAACAGC (SEQ ID NO: 100) MCM66 CCAGGCAAATTCTGTTTTATCAG(SEQ ID NO: 101) EL1000 GCACTGTCTTTCCGTCTGCTGC (SEQ ID NO: 102) MCM165GCGAACGATGCATAAAGGAGGTAAAAAAACATGGTATCCTGTTC TGCGCCGGGTAAGATTTACCTG(SEQ ID NO: 103) MCM177 GGGCCCGTTTAAACTTTAACTAGACTTTAATCTACTTTCAGACCTTGC (SEQ ID NO: 104) EL1003 GATAGTAACGGCTGCGCTGCTACC (SEQ ID NO: 105)EL1006 GACAGCTTATCATCGACTGCACG (SEQ ID NO: 106) MCM161CACCATGGTATCCTGTTCTGCG (SEQ ID NO: 107) MCM162 TTAATCTACTTTCAGACCTTGC(SEQ ID NO: 108)II. Construction of MCM442-449: BL21 and BL21(DE3) withFRT-cmR-FRT-gi1.x-mKKDyIi) Construction of Template for Recombination

FRT-based recombination cassettes, and plasmids for Red/ET-mediatedintegration and antibiotic marker loopout were obtained from GeneBridges GmbH (Germany). Procedures using these materials were carriedout according to Gene Bridges protocols. Primers MCM193 and MCM195 wereused to amplify the resistance cassette from the FRT-gb2-Cm-FRT templateusing Stratagene Herculase II Fusion kit according to the manufacturer'sprotocol. The 50 uL reaction was cycled as follows: 95° C., 2 minutes;(95° C., 20 seconds, 55° C., 20 seconds, 72° C., 1 minute)×5, (95° C.,20 seconds, 60° C., 20 seconds, 72° C., 1 minute)×25; 72° C., 3 minutes;4° C. until cool. The amplicon was purified by a Qiagen PCR columnaccording to the manufacturer's protocol and eluted in 30 uL EB (ElutionBuffer). DNA was digested with NdeI and PciI in a 20 uL reaction with 1×Roche H buffer and 0.5 uL BSA. Plasmid MCM376 was digested in a 10 uLreaction containing 1 uL each of NdeI, NcoI, and Roche H buffer.Reactions proceeded overnight at 37° C., and then cut DNA was purifiedon Qiagen PCR columns and eluted in 30 uL EB. The PCR product wasligated into MCM376 in a reaction containing 1 uL vector, 3 uL PCRproduct, 1 uL Roche Quick Ligase Buffer 2, 5 uL Buffer 1, and 1 uLLigase. The reaction proceeded at room temperature for 3 hours and then5 uL was transformed into Invitrogen TOP10 cells according to themanufacturer's protocol. Transformants were selected on L agar (LA) andchloramphenicol (10 ug/mLO) at 37° C. overnight.

Transformant colonies were patched onto LA containing chloramphenicol(30 ug/mL) and kanamycin (50 ug/ml) for storage and sent to Quintara(Berkeley, Calif.) for sequencing. Four clones, one each with the fourdifferent nucleotides at the “N” in primer MCM195, were found to havethe correct sequence for the inserted promoter. Clones were grown in 5mL LB containing chloramphenicol (30 ug/mL) and kanamycin (50 ug/mL) andused for the preparation of plasmid DNA. This plasmid was retransformedinto TOP10 cells and strains were frozen as:

TABLE 5 MCM484-487 MCM484 cmR-gi1.6-MVK(mazei) in pET (clone A1-3,variable nt A) MCM485 cmR-gi1.0-MVK(mazei) in pET (clone B4-6, variablent C) MCM486 cmR-gi1.2-MVK(mazei) in pET (clone C1-5, variable nt G)MCM487 cmR-gi1.5-MVK(mazei) in pET (clone C3-3, variable nt T)ii) Creation of Recombination Target Strains MCM349 and MCM441

The chloramphenicol resistance (cmR) marker was looped out of strainMCM331 using plasmid pGB706 (GeneBridges) according to Manufacturer'sinstructions. MCM331 cells were grown to mid-log in LB and washed threetimes in iced, sterile water. A 1 uL aliquot of pGB706 DNA was added to50 uL of cell suspension and this mixture was electroporated in a 2 mmcuvette at 2.5 volts, 25 uFd followed immediately by recovery in 500 uLLB for one hour at 30 C. Transformants were selected on LB containingtetracycline (5 ug/ml) at 30° C. The following day, a clone was grown upat 30° C. in LB containing tetracycline (5 ug/ml) until visibly turbid(OD₆₀₀-0.5-0.8). This culture was streaked onto LB and grown overnightat 37° C. A clone that was unable to grow on LB containingchloramphenicol (10 ug/mL) or LB containing tetracycline (5 ug/mL) wasfrozen as MCM348. Plasmid MCM356 (pRedET carbencillin; GeneBridges) waselectroporated in as described above and transformants were selected onLB containing carbenicillin (50 ug/mL) at 30° C. A clone was grown in LBcarbenicillin (50 ug/mL) at 30° C. and frozen as MCM349.

Strain MCM441 was created by electrotransforming plasmid MCM356 intoEWL204 as above.

iii) Recombination of FRT-cmR-FRT-gi1.x-mMVK into MCM349 and MCM441

Plasmids MCM484-487 were used as template for PCR amplification withprimers MCM120 and MCM196 and Herculase II Fusion kit, according to themanufacturer's protocol. Three reactions per template were carried out,with 0, 1, or 3 uL DMSO. The 50 uL reactions were cycled as follows: 95°C., 2 minutes; (95° C., 20 seconds; 55° C. 20 seconds; 72° C., 1.5minutes) for five cycles; (95° C., 20 seconds; 60° C. 20 seconds; 72°C., 1.5 minutes) for 25 cycles; 72° C. for 3 minutes; 4° C., overnight.]The three reactions from a given template were pooled and purified onQiagen PCR columns and eluted with 30 uL EB at 60° C. 5 uL DNA wasdigested with 1 uL DpnI in 1× Roche Buffer A for 3 hours at 37° C. ThisDNA was then microdialyzed against excess water for 30 minutes.

Strains were grown in 5 mL LB containing carbenicillin (50 ug/mL) fromfresh streaks at 30 C to an OD600 of ˜0.5. 40 mM L-arabinose was addedand cultures were incubated at 37 C for 1.5 hours. Cells were harvestedand electroporated with 3 uL dialyzed amplicons above, and thenrecovered in 500 uL SOC at 37 C for 1.5-3 hours. Transformants wereselected on LA plates containing chloramphenicol (5 ug/mL) at 37° C.

Kanamycin sensitive clones were screened by PCR for insertion of theamplicon. PCR products from positive clones were sequenced to verify thesequence of inserted DNA. Amplicons were consistent with theFRT-gi1.2-yKKDyI at attTn7 in MCM441 and 348 being replaced byFRT-cmR-FRT-gi1.x-mKKDyI (The yK and mK designations refer to themevalonate kinase from Saccharomyces cerevisiae and Methanosarcina mazeirespectively).

TABLE 6A The following strains were grown in LB containingchloramphenicol (5 ug/mL) and frozen. Recombi- nation Amplicon Strain IDName Parent Template MCM442 BL21(DE3) cmR-gi1.6mKKDyI A1, MCM349 MCM484clone37 (A) MCM443 BL21(DE3) cmR-gi1.0mKKDyI B4, MCM349 MCM485 clone27(C) MCM444 BL21(DE3) cmR-gi1.2mKKDyI C1, MCM349 MCM486 clone16 (G)MCM445 BL21(DE3) cmR-gi1.5mKKDyI C3, MCM349 MCM487 clone7 (T) MCM446BL21 cmR-gi1.6mKKDyI A1-3 (A) MCM441 MCM484 MCM447 BL21 cmR-gi1.0mKKDyIB4-6 (C) MCM441 MCM485 MCM448 BL21 cmR-gi1.2mKKDyI C1-5 (G) MCM441MCM486 MCM449 BL21 cmR-gi1.5mKKDyI C3-3 (T) MCM441 MCM487

TABLE 6B Primers MCM120 AAAGTAGCCGAAGATGACGGTTTGTCACATGGAGTTGGCAGGATGTTTGATTAAAAGCAATTAACCCTCACTAAAGGGCGG (SEQ ID NO: 109) MCM193GATATACATATGAATTAACCCTCACTAAAGG (SEQ ID NO: 110) MCM195GCATGCATGACATGTTTTTTTACCTCCTTTGTTATCCGCTCACAATTAGTGGTTGAATTATTTGCTCAGGATGTGGCATNGTCAAGGGCGCGGCCGCGATCTAATACGACTCACTATAGGGCTCG (SEQ ID NO: 111) MCM196AGGCTCTCAACTCTGACATGTTTTTTTCCTCCTTAAGGGTGCAGGCCTATCGCAAATTAGCTTAATCTACTTTCAGACCTTGCTCGG (SEQ ID NO: 112)III. The Effect of Yeast Extract on Isoprene Production in E. coliExpressing Genes from the Mevalonic Acid Pathway and Grown in Fed-BatchCulture at the 15-L Scale

Medium Recipe (Per Liter Fermentation Medium):

K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferricammonium citrate 0.3 g, yeast extract 0.5 g, 1000× Modified Trace MetalSolution 1 ml. All of the components were added together and dissolvedin diH₂O. This solution was autoclaved. The pH was adjusted to 7.0 withammonium hydroxide (30%) and q.s. to volume. Glucose 10 g, thiamine*HCl0.1 g, and antibiotics were added after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

Citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g,CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg,NaMoO₄*2H₂O 100 mg. Each component is dissolved one at a time in Di H₂O,pH to 3.0 with HCl/NaOH, then q.s. to volume and filter sterilized witha 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the upper mevalonic acid (MVA) pathway (pCL Upper), theintegrated lower MVA pathway (gi1.2KKDyI), and high expression ofmevalonate kinase from M. mazei and isoprene synthase from P. alba(pTrcAlba-mMVK). This experiment was carried out to monitor isopreneformation from glucose at the desired fermentation pH 7.0 andtemperature 30° C. A frozen vial of the E. coli strain was thawed andinoculated into tryptone-yeast extract medium. After the inoculum grewto OD 1.0, measured at 550 nm, 500 mL was used to inoculate a 15-Lbioreactor bringing the initial volume to 5-L.

i) Production of Isoprene in E. coli Cells (EL256) Grown in Fed-BatchCulture Without Yeast Extract Feeding

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. The total amount of glucose delivered to thebioreactor during the 67 hour fermentation was 3.9 kg. Induction wasachieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG). TheIPTG concentration was brought to 102 uM when the optical density at 550nm (OD₅₅₀) reached a value of 9. The IPTG concentration was raised to192 uM when OD₅₅₀ reached 140. The OD₅₅₀ profile within the bioreactorover time is shown in FIG. 67A. The isoprene level in the off gas fromthe bioreactor was determined using a Hiden mass spectrometer. Theisoprene titer increased over the course of the fermentation to a finalvalue of 35.6 g/L (FIG. 67B). The total amount of isoprene producedduring the 67 hour fermentation was 320.6 g and the time course ofproduction is shown in FIG. 67C. The metabolic activity profile, asmeasured by TCER, is shown in FIG. 67D. The molar yield of utilizedcarbon that went into producing isoprene during fermentation was 17.9%.The weight percent yield of isoprene from glucose was 8.1%.

Production of Isoprene in E. coli Cells (EL256) Grown in Fed-BatchCulture with Yeast Extract Feeding

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. The total amount of glucose delivered to thebioreactor during the 68 hour fermentation was 7.1 kg. A total of 1.06kg of yeast extract was also fed during the fermentation. Induction wasachieved by adding IPTG. The IPTG concentration was brought to 208 uMwhen the optical density at 550 nm (OD₅₅₀) reached a value of 7. TheIPTG concentration was raised to 193 uM when OD₅₅₀ reached 180. TheOD₅₅₀ profile within the bioreactor over time is shown in FIG. 68A. Theisoprene level in the off gas from the bioreactor was determined using aHiden mass spectrometer. The isoprene titer increased over the course ofthe fermentation to a maximum value of 32.2 g/L (FIG. 68B). The totalamount of isoprene produced during the 68 hour fermentation was 395.5 gand the time course of production is shown in FIG. 68C. The time courseof volumetric productivity is shown in FIG. 68D and shows that anaverage rate of 1.1 g/L/hr was maintained for between 23 and 63 hours.The metabolic activity profile, as measured by CER, is shown in FIG. 68EThe molar yield of utilized carbon that went into producing isopreneduring fermentation was 10.3%. The weight percent yield of isoprene fromglucose was 5.2%.

IV. Production of Isoprene from Different Carbon Sources in E. coliHarboring the Mevalonic Acid (MVA) Pathway and Isoprene Synthase(EWL256)

Media Recipe (Per Liter Fermentation Media):

K₂HPO₄ 13.6 g, KH₂PO₄ 13.6 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2g, ferric ammonium citrate 0.3 g, (NH₄)₂SO₄ 3.2 g, yeast extract 0.2 g,1000× Modified Trace Metal Solution 1 ml. All of the components weredissolved sequentially in diH₂O. The pH was adjusted to 6.8 withammonium hydroxide (30%) and brought to volume. Media was filtersterilized with a 0.22 micron filter. Carbon source was added to a finalconcentration of 1%. Required antibiotics were added after sterilizationand pH adjustment.

1000× Trace Metal Solution (Per Liter Fermentation Media):

Citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g,CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg,NaMoO₄*2H₂O 100 mg. Each component was dissolved one at a time in diH₂O,pH to 3.0 with HCl/NaOH, and then brought to volume and filtersterilized with a 0.22 micron filter.

i) Preparation of AFEX Biomass Hydrolysate

AFEX pretreated corn stover was hydrolyzed to prepare biomasshydrolysate containing both xylose, glucose and acetate.

AFEX pretreated corn stover, received from Michigan BiotechnologyInstitute, was used. The pretreatment conditions were, 60% moisture, 1:1ammonia loading, and 90° C. for 30 minutes, then air dried. The moisturecontent in the AFEX pretreated corn stover was 21.27%. Content of glucanand xylan in the AFEX pretreated corn stover were 31.7% and 19.1% (drybasis) respectively. The enzyme used was accellerase 1000, GrindamylH121 (Danisco xylanase product from Aspergillus niger for bread-makingindustry).

For saccharification, 20 g of AFEX pretreated corn stover was added intoa 500 ml flask, together with 5 ml of 1 M pH 4.8 sodium citrate buffer,2.25 ml of Accellerase 1000, 0.1 ml of Grindamyl H121, and 72.65 ml ofDI water. The flask was put in an orbital shaker, and incubated at 50°C. for 96 hours.

For analysis, one sample was taken from the shaker, and analyzed usingHPLC. The hydrolysate contained 37.2 g/l of glucose and 24.3 g/L ofxylose, and 7.6 g/L of oligomers of glucose and/or xylose. Additionally,the hydrolysate also contains 1.17 g/L acetate.

ii) Experimental Procedure

An inoculum of the E. coli strain EWL256 containing the MVA pathway andisoprene synthase was taken from a frozen vial and streaked onto an LBbroth agar plate containing spectinomycin (50 ug/mL) and carbinicllin(50 ug/mL) and incubated at 30° C. overnight. A single colony wasinoculated into TM3 media containing glucose, xylose, glycerol, acetateor biomass as only carbon source and grown overnight at 30° C. Cellsgrow on acetate reached a significantly lower optical density. Cellsgrown on glucose, glycerol, biomass hydrolysate or acetate were dilutedinto 20 mL of TM3 media containing the respective carbon sources toreach an optical density of between 0.1 measured at 600 nM. A negativecontrol not containing any carbon source was prepared from the glucoseovernight culture. A separate experiment was performed with glucose andxylose, where the cultures were diluted to an optical density of 0.05.All culture conditions (except for acetate and glycerol) were tested induplicates and the presented results are averaged between thesecultures. Production of isoprene was induced with 200 μM IPTG from thebeginning of the experiment. The flasks were incubated at 30° C. in anorbital shaker (200 rpm) and growth was followed by measuring opticaldensity. After the glucose fed cultures had reached an optical densityof approximately 0.4, samples were analyzed for isoprene production fromall the tested carbon sources every hour for three hours. Samples of 100μL were transferred in duplicates to 2 mL glass vials, sealed andincubated for 30 min at 30° C. The bacteria were then heat killed byincubation at 80° C. for 8 minutes. The amount of produced isoprene wasmeasured using GC-MS and specific productivity (μg/L*hr) was calculated.

iii) Results

Significant production of isoprene could be demonstrated during growthon all the tested carbon sources. These carbon sources are examples ofcommon alcohols, organic acids, sugars containing 5 or 6 carbon units(C5 or C6), and biomass hydrolysate.

The initial growth rate on biomass hydrolysate was comparable to thegrowth rate on glucose (FIG. 69A). The initial specific productivityduring growth on biomass hydrolysate was significantly higher thanduring growth on glucose. This demonstrates that biomass hydrolysate canbe used as an efficient source of carbon for the production of isoprene.The specific productivity declined after 255 minutes of growth onbiomass hydrolysate (FIG. 69B). The bacteria had a slower growth ratewith xylose as only carbon source when compared to glucose (FIG. 69C),but a significant specific isoprene productivity was measured (FIG.69D). This shows that both C5 and C6 sugars can be utilized for theproduction of isoprene via the mevalonate acid pathway.

Surprisingly, bacteria grown on acetate as the only carbon source had aspecific productivity of isoprene approximately twice as high as duringgrowth on glucose (FIG. 69A). The bacteria grew slower on acetate whencompared to glucose (FIG. 69B), but the performed experimentdemonstrates that acetate can also be used as a carbon source for theproduction of isoprene. Acetate was also present in the biomasshydrolysate as measured by HPLC.

The bacteria grew well with glycerol as only carbon source (FIG. 69A)and significant production of isoprene was demonstrated (FIG. 69B). Thisshows that common alcohols may also be used as carbon sources forproduction of isoprene via the mevalonate acid pathway.

Example 11 Expression of Isoprene-Synthase from Plant in Streptomycessp.

The gene for isoprene synthase Kudzu was obtained from plasmidpJ201:19813. Plasmid pJ201:19813 encodes isoprene synthase from Pueraialobata (Kudzu plant) and was codon-optimized for Pseudomonasfluorescens, Pseudomonas putida, Rhodopseudomonas palustris andCorynebacterium (FIGS. 79A-79C (SEQ ID NO:123)). Digestion of plasmidpJ201:19813 with restriction enzymes NdeI and BamHI liberated geneiso19813 that was ligated into the Streptomyces-E. coli shuttle vectorpUWL201PW (Doumith et al., Mol. Gen. Genet. 264: 477-485, 2000; FIG. 71)to generate pUWL201_iso. Successful cloning was verified by restrictionanalysis of pUWL201_iso. Expression of isoprene synthase iso19813 wasunder control of the erm-promoter which allows for constitutiveexpression in Streptomycetes species, but not for expression in E. coli.

PUWL201PW (no insert) and pUWL201_iso were introduced in Streptomycesalbus J1074 (Sanchez et al., Chem. Biol. 9:519-531, 2002) bytransformation of protoplasts as described by Hopwood et al., The Johninnes foundation, Norwich, 1985.

A 200 μl aliquot of protoplast suspensions was transformed with 1.9 μgpUWL201PW or 2.9 μg pUWL201_iso. After incubation overnight at 28° C. onnon-selective R5-agarplates, positive transformants were selected byfurther incubation for 4 days in R3-overlay agar containing thiostrepton(250 μg/ml). Thiostrepton resistant transformants were examined forpresence of the pUWL-plasmids by plasmid preparation using Plasmid MiniKit (Qiagen). Prepared plasmid DNA was reintroduced in E. coli DH5α togenerate sufficient amounts of plasmid DNA to be analyzed by restrictionanalysis. Positive transformants were selected on ampicillin-containingL-agar plates and insert analysis was done by digestion of plasmid DNAwith NdeI and BamHI endonucleases. Isoprene synthase was identified as a1.7 kb fragment in positive pUWL201 iso clones while in the controlstrains (pUWL201PW) no such fragment was observed.

Wild type strain and transformants of S. albus containing controlplasmid pUWL201PW or isoprene synthase encoding pUWL201_iso wereanalyzed for isoprene formation. Strains were cultivated in duplicate onsolid media (tryptic soy broth agar, TSB; 2.5 ml) in presence or absenceof thiostrepton (200 μg/ml) and incubated for 4 days at 28° C. in sealedhead-space vials (total volume 20 ml). 500 μl head-space samples (endpoint measurements) were analyzed by GC-MS in SIM-mode and isoprene wasidentified according to reference retention times and molecular masses(67 m/z). Isoprene present in head-space samples was quantified bypreviously generated calibration curves. While wild-type S. albus andcontrol strains harboring pUWL201PW produced isoprene in concentrationsslightly higher than the detection limit (0.04-0.07 ppm), S. albusharboring pUWL201_iso produced isoprene in at least tenfold excesscompared to controls (0.75 ppm; FIG. 72). The results demonstratesuccessful expression of plant-derived isoprene synthase in aprokaryotic organism of the Actinomycetes group.

Example 12 Production of Isoprene or Mevalonate from Fatty Acid or PalmOil in E. coli fadR atoC LS5218 Containing the Upper or Upper and LowerMevalonic Acid Pathway Plus Kudzu Isoprene Synthase

Escherichia coli fadR atoC strain LS5218 (#6966) was obtained from theColi Genetic Stock Center. FadR encodes a transcription repressor thatnegatively regulates expression of the genes encoding fatty aciddegradation enzymes (Campbell et al., J. Bacteriol. 183: 5982-5990,2001). AtoC is a response regulator in a two-component regulatory systemwith AtoS, regulates acetolactate metabolism. The fadR atoC strainallows constitutive expression of the fatty acid degradation genes andincorporates long chain fatty acids into long-chain-lengthpolyhydroxyalkanoates. When palm oil is used as a carbon source foreither mevalonate or isoprene production, the palm oil was converted toglycerol plus fatty acid. Methods for this are well known in the art,and it can be done either enzymatically by incubation with a lipase (forexample Porcine pancreatic lipase, Candida rugosa lipase, or othersimilar lipases) or chemically by saponification with a base such assodium hydroxide.

i) E. coli fadR atoC Strain Expressing the Upper Mevalonic Acid Pathway

Strain WW4 was created by electroporating pCLPtrcUpperPathway intoLS5218 using standard methods (Sambrooke et al., Molecular Cloning: ALaboratory Manual, 2^(nd) ed., Cold Spring Harbor, 1989). Incorporationof the plasmid was demonstrated by the production of mevalonic acid(MVA) when cells were cultured in TM3 medium supplemented with eitherC12 fatty acid (FA) or palm oil as the carbon source. To demonstrateproduction of MVA by WW4 from fatty acid, cells from an overnightculture were diluted 1 to 100 into 5 mL of modified TM3 medium (TM3without yeast extract) supplemented with 0.25% C12 FA (Sigma cat#L9755). The first sign of MVA production (24 mg/L) was apparent afterovernight incubation at 30° C. of the IPTG induced culture. Productionincreased over three days with the final level of 194 mg/L of MVAproduced. To demonstrate production of MVA by WW4 from oil, cells froman overnight culture were diluted 1 to 100 into modified TM3 mediumsupplemented with 200 mg of digested palm oil per 5 mL of TM3 medium.The first sign of MVA production (50 mg/L) was apparent after overnightincubation of the IPTG induced culture at 30° C. Production increasedover three days with a final level of 500 mg/L of MVA produced.

ii) E. coli fadR atoC Strain Expressing the Upper and Lower MVA PathwayPlus Kudzu Isoprene Synthase

Escherichia coli strain WW4 (LS5218 fadR atoC pCLPtrcUpperPathway) wastransformed with pMCM118 [pTrcKKDyIkIS] to yield WW10. The incorporationof the plasmid was demonstrated by evidence of production of isoprenewhen the strain was cultured in TM3 and glucose and induced with IPTG(100, 300, or 900 uM). The strain was relatively sensitive to IPTG andshowed a significant growth defect even at 100 uM IPTG. These resultsare shown in FIG. 70A.

To test isoprene production from dodecanoic acid, WW10 was culturedovernight in L broth containing spectinomycin (50 ug/ml), and kanamycin(50 ug/ml) at 37 C with shaking at 200 rpm. The cells were washed withmodified TM3 medium by centrifugation and resuspension in their originalculture volume with this medium. The washed and resuspended cells fromthis starter culture were diluted 1 to 100 and 1 to 10 into 5 mL ofmodified TM3 medium containing 0.125% C12 Fatty Acid (Sigma cat #L9755).

To demonstrate production of mevalonate from palm oil, the oil waspredigested with lipase at 37° C. and 250 rpm for several days torelease the fatty acids (evidence of hydrolysis was judged by the foamformed when tubes were shaken).

In addition, a culture was set up by diluting the washed cells at 1 to10 into modified TM3 medium contained in test tubes with palm oil. Afurther tube was set up by the addition of 0.125% C12FA to the remainder(2.5 mL) of the washed cells without further dilution (bioconversion).After 3.75 hours of growth at 30° C. with shaking at 250 rpm all of thecultures were induced by the addition of 50 uM IPTG. Incubation wascontinued for 4 hours after which time 200 uL of each of the cultureswas assayed for isoprene accumulation with a modified head space assay(1 hour accumulation at 30° C. with shaking at 500 rpm). An additionalisoprene assay was conducted by a 12 hour incubation of the assay glassblock prior to GCMS analysis. Incubation of the induced cultures wascontinued overnight and 200 uL aliquots were again assayed for isopreneproduction (1 hour, 30 deg, 500 rpm Shel-Lab shaker) the followingmorning. Analysis of these cultures showed the production of significantlevels of isoprene. The highest levels of isoprene were observed in theculture which was seeded at 1/10 dilution from the overnight starterculture after it had been incubated and induced overnight. This resultsuggests that this culture continued to grow and increase in celldensity. These results are shown in FIG. 70B. Cell density could not bemeasured directly because the fatty acid suspension had a turbidappearance. Cell density of this culture was therefore determined byplating an aliquot of the culture and showed 8×10⁷ colony forming units.This corresponds approximately to an OD₆₀₀ of 0.1. Nevertheless, thisculture provided significant isoprene production; no isoprene isobserved for similar strains without the pathway described in thisexample.

Example 13 Improvement of Isoprene Production by Constitutive Expressionof ybhE in E. coli

This example shows production of isoprene in a strain constitutivelyexpressing ybhE (pgl) compared to a control strain with wild type ybhE.The gene ybhE (pgl) encodes a 6-phosphogluconolactonase that suppressesposttranslational gluconylation of heterologously expressed proteins andimproves product solubility and yield while also improving biomass yieldand flux through the pentose phosphate pathway (Aon et al. Applied andEnvironmental Microbiology, 74(4): 950-958, 2008).

The BL21 strain of E. coli producing isoprene (EWL256) was constructedwith constitutive expression of the ybhE gene on a replicating plasmidpBBR1MCS5 (Gentamycin) (obtained from Dr. K. Peterson, Louisiana StateUniversity).

FRT-based recombination cassettes, and plasmids for Red/ET-mediatedintegration and antibiotic marker loopout were obtained from GeneBridges GmbH (Germany). Procedures using these materials were carriedout according to Gene Bridges protocols. Primers Pgl-F and PglGI1.5-Rwere used to amplify the resistance cassette from the FRT-gb2-Cm-FRTtemplate using Stratagene Herculase II Fusion kit according to themanufacturer's protocol. The PCR reaction (50 uL final volume)contained: 5 uL buffer, 1 uL template DNA (FRT-gb2-Cm-F from GeneBridges), 10 pmols of each primer, and 1.5 uL 25 mM dNTP mix, made to 50uL with dH₂O. The reaction was cycled as follows: 1×2 minutes, 95° C.then 30 cycles of (30 seconds at 95° C.; 30 seconds at 63° C.; 3 minutesat 72° C.).

The resulting PCR product was purified using the QiaQick PCRpurification kit (Qiagen) and electroporated into electrocompetentMG1655 cells harboring the pRed-ET recombinase-containing plasmid asfollows. Cells were prepared by growing in 5 mLs of L broth to andOD600˜0.6 at 30° C. The cells were induced for recombinase expression bythe addition of 4% arabinose and allowed to grow for 30 minutes at 30°C. followed by 30 minutes of growth at 37° C. An aliquot of 1.5 mLs ofthe cells was washed 3-4 times in ice cold dH₂O. The final cell pelletwas resuspended in 40 uL of ice cold dH₂O and 2-5 uL of the PCR productwas added. The electroporation was carried out in 1-mm gap cuvettes, at1.3 kV in a Gene Pulser Electroporator (Bio-Rad Inc.). Cells wererecovered for 1-2 hours at 30° C. and plated on L agar containingchloramphenicol (5 ug/mL). Five transformants were analyzed by PCR andsequencing using primers flanking the integration site (2 primer sets:pgl and 49 rev and 3′ EcoRV-pglstop; Bottom Pgb2 and Top GB's CMP(946)). A correct transformant was selected and this strain wasdesignated MG1655 GI1.5-pgl::CMP.

The chromosomal DNA of MG1655 GI1.5-pgl::CMP was used as template togenerate a PCR fragment containing the FRT-CMP-FRT-GI1.5-ybhE construct.This construct was cloned into pBBR1MCS5(Gentamycin) as follows. Thefragment, here on referred to as CMP-GI1.5-pgl, was amplified using the5′ primer Pglconfirm-F and 3′ primer 3′ EcoRV-pglstop. The resultingfragment was cloned using the Invitrogen TOPO-Blunt cloning kit into theplasmid vector pCR-Blunt II-TOPO as suggested from the manufacturer. TheNsiI fragment harboring the CMP-GI1.5-pgl fragment was cloned into thePstI site of pBBR1MCS5(Gentamycin). A 20 μl ligation reaction wasprepared containing 5 μl CMP-GI1.5-pgl insert, 2 μl pBBR1MCS5(Gentamycin) vector, 1 μl T4 DNA ligase (New England Biolabs), 2 μl 10×ligase buffer, and 10 μl ddH₂O. The ligation mixture was incubated atroom temperature for 40 minutes then 2-4 uL were electroporated intoelectrocompetent Top10 cells (Invitrogen) using the parameters disclosedabove. Transformants were selected on L agar containing 10 ug/mlchloramphenicol and 5 ug/ml Gentamycin. The sequence of the selectedclone was determined using a number of the primers described above aswell as with the in-house T3 and Reverse primers provided by Sequetech,CA. This plasmid was designated pBBRCMPGI1.5-pgl (FIGS. 77A-B and SEQ IDNO:122).

Plasmid pBBRCMPGI1.5-pgl was electroporated into EWL256, as describedabove in Example 10 and transformants were plated on L agar containingChloramphenicol (10 ug/mL), Gentamycin (5 ug/mL), spectinomycin (50ug/mL), and carbenicillin (50 ug/mL). One transformant was selected anddesignated RM11608-2.

Primers:

Pgl-F (SEQ ID NO: 115) 5′-ACCGCCAAAAGCGACTAATTTTAGCTGTTACAGTCAGTTGAATTAACCCT CACTAAAGGGCGGCCGC-3′PglGI1.5-R (SEQ ID NO: 116) 5′-GCTGGCGATATAAACTGTTTGCTTCATGAATGCTCCTTTGGGTTACCTCCGGGAAACGCGGTTGATTTGTTTAGTGGTTGAATTATTTGCTCAGGATGTGGCATAGTCAAGGGCGTGACGGCTCGCTAATACGACTCACTATAGGGCTCG AG-3′ 3′EcoRV-pglstop: (SEQ ID NO: 117)5′-CTT GAT ATC TTA GTG TGC GTT AAC CAC CAC pgl +49 rev: (SEQ ID NO: 118)CGTGAATTTGCTGGCTCTCAG Bottom Pgb2: (SEQ ID NO: 119)GGTTTAGTTCCTCACCTTGTC Top GB's CMP (946): (SEQ ID NO: 120)ACTGAAACGTTTTCATCGCTC Pglconfirm-F (SEQ ID NO: 121)5′-ACCGCCAAAAGCGACTAATTTTAGCT-3′i) Small Scale Analysis

Media Recipe (Per Liter Fermentation Media):

K₂HPO₄ 13.6 g, KH₂PO₄ 13.6 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2g, ferric ammonium citrate 0.3 g, (NH₄)₂SO₄ 3.2 g, yeast extract 1 g,1000× Trace Metals Solution 1 ml. All of the components were addedtogether and dissolved in diH₂O. The pH was adjusted to 6.8 withammonium hydroxide (30%) and brought to volume. Media wasfilter-sterilized with a 0.22 micron filter. Glucose 5.0 g andantibiotics were added after sterilization and pH adjustment.

1000× Trace Metal Solution (Per Liter Fermentation Media):

Citric Acid*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g,CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg,NaMoO₄*2H₂O 100 mg. Each component is dissolved one at a time in diH₂O.The pH is adjusted to 3.0 with HCl/NaOH, and then the solution isbrought to volume and filter-sterilized with a 0.22 micron filter.

a) Experimental Procedure

Isoprene production was analyzed by growing the strains in a Cellerator™from MicroReactor Technologies, Inc. The working volume in each of the24 wells was 4.5 mL. The temperature was maintained at 30° C., the pHsetpoint was 7.0, the oxygen flow setpoint was 20 sccm and the agitationrate was 800 rpm. An inoculum of E. coli strain taken from a frozen vialwas streaked onto an LB broth agar plate (with antibiotics) andincubated at 30° C. A single colony was inoculated into media withantibiotics and grown overnight. The bacteria were diluted into 4.5 mLof media with antibiotics to reach an optical density of 0.05 measuredat 550 nm.

Off-gas analysis of isoprene was performed using a gaschromatograph-mass spectrometer (GC-MS) (Agilent) headspace assay.Sample preparation was as follows: 100 μL of whole broth was placed in asealed GC vial and incubated at 30° C. for a fixed time of 30 minutes.Following a heat kill step, consisting of incubation at 70° C. for 5minutes, the sample was loaded on the GC.

Optical density (OD) at a wavelength of 550 nm was obtained using amicroplate reader (Spectramax) during the course of the run. Specificproductivity was obtained by dividing the isoprene concentration (μg/L)by the OD reading and the time (hour).

The two strains EWL256 and RM11608-2 were assessed at 200 and 400 uMIPTG induction levels. Samples were analyzed for isoprene production andcell growth (OD550) at 1, 2.5, 4.75, and 8 hours post-induction. Sampleswere done in duplicate.

b) Results

The experiment demonstrated that at 2 different concentrations of IPTGthe strain expressing the ybhE (pgl) had a dramatic 2-3 fold increase inspecific productivity of isoprene compared to the control strain.

ii) Isoprene Fermentation from E. coli Expressing M. mazei MevalonateKinase, P. alba Isoprene Synthase, and pgl Over-Expression (RHM111608-2)and Grown in Fed-Batch Culture at the 15-L Scale

Medium Recipe (Per Liter Fermentation Medium)

K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferricammonium citrate 0.3 g, yeast extract 0.5 g, 1000× Modified Trace MetalSolution 1 ml. All of the components were added together and dissolvedin diH₂O. This solution was autoclaved. The pH was adjusted to 7.0 withammonium hydroxide (30%) and q.s. to volume. Glucose 10 g, thiamine*HCl0.1 g, and antibiotics were added after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

Citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g,CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg,NaMoO₄*2H₂O 100 mg. Each component is dissolved one at a time in Di H₂O,pH to 3.0 with HCl/NaOH, then q.s. to volume and filter sterilized witha 0.22 micron filter

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the upper mevalonic acid (MVA) pathway (pCL Upper), theintegrated lower MVA pathway (gi1.2KKDyI), high expression of mevalonatekinase from M. mazei and isoprene synthase from P. alba (pTrcAlba-mMVK),and high expression of pgl (pBBR-pgl). This experiment was carried outto monitor isoprene formation from glucose at the desired fermentationpH 7.0 and temperature 34° C. A frozen vial of the E. coli strain wasthawed and inoculated into tryptone-yeast extract medium. After theinoculum grew to OD 1.0, measured at 550 nm, 500 mL was used toinoculate a 15-L bioreactor bringing the initial volume to 5-L.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. The total amount of glucose delivered to thebioreactor during the 40 hour (59 hour) fermentation was 3.1 kg (4.2 kgat 59 hour). Induction was achieved by adding IPTG. The IPTGconcentration was brought to 110 uM when the optical density at 550 nm(OD₅₅₀) reached a value of 4. The IPTG concentration was raised to 192uM when OD₅₅₀ reached 150. The OD₅₅₀ profile within the bioreactor overtime is shown in FIG. 78A. The isoprene level in the off gas from thebioreactor was determined using a Hiden mass spectrometer. The isoprenetiter increased over the course of the fermentation to a maximum valueof 33.2 g/L at 40 hours (48.6 g/L at 59 hours) (FIG. 78B). The isoprenetiter increased over the course of the fermentation to a maximum valueof 40.0 g/L at 40 hours (60.5 g/L at 59 hours) (FIG. 78C). The totalamount of isoprene produced during the 40-hour (59-hour) fermentationwas 281.3 g (451.0 g at 59 hours) and the time course of production isshown in FIG. 78D. The time course of volumetric productivity is shownin FIG. 78E and shows that an average rate of 1.0 g/L/hr was maintainedbetween 0 and 40 hours (1.4 g/L/hour between 19 and 59 hour). Themetabolic activity profile, as measured by CER, is shown in FIG. 78F.The molar yield of utilized carbon that went into producing isopreneduring fermentation was 19.6% at 40 hours (23.6% at 59 hours). Theweight percent yield of isoprene from glucose was 8.9% at 40 hours(10.7% at 59 hours).

Unless defined otherwise, the meanings of all technical and scientificterms used herein are those commonly understood by one of skill in theart to which this invention belongs. Singleton, et al., Dictionary ofMicrobiology and Molecular Biology, 2nd ed., John Wiley and Sons, NewYork (1994), and Hale & Marham, The Harper Collins Dictionary ofBiology, Harper Perennial, N.Y. (1991) provide one of skill with ageneral dictionary of many of the terms used in this invention. It is tobe understood that this invention is not limited to the particularmethodology, protocols, and reagents described, as these may vary. Oneof skill in the art will also appreciate that any methods and materialssimilar or equivalent to those described herein can also be used topractice or test the invention.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention which can be had by reference to thespecification as a whole.

For use herein, unless clearly indicated otherwise, use of the terms“a”, “an,” and the like refers to one or more.

Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X.” Numeric ranges are inclusive of the numbers defining the range.

It is understood that aspects and embodiments of the invention describedherein include “comprising,” “consisting,” and “consisting essentiallyof” aspects and embodiments.

Appendix 1

Exemplary 1-deoxy-D-xylulose-5-phosphate Synthase Nucleic Acids andPolypeptides

-   ATH: AT3G21500(DXPS1) AT4G15560(CLA1) AT5G11380(DXPS3)-   OSA: 4338768 4340090 4342614-   CME: CMF089C-   PFA: MAL13P1.186-   TAN: TA20470-   TPV: TP01_(—)0516-   ECO: b0420(dxs)-   ECJ: JW0410(dxs)-   ECE: Z0523(dxs)-   ECS: ECs0474-   ECC: c0531(dxs)-   ECI: UTI89_C0443(dxs)-   ECP: ECP_(—)0479-   ECV: APECO1_(—)1590(dxs)-   ECW: EcE24377A_(—)0451(dxs)-   ECX: EcHS_A0491-   STY: STY0461(dxs)-   STT: t2441(dxs)-   SPT: SPA2301(dxs)-   SEC: SC0463(dxs)-   STM: STM0422(dxs)-   YPE: YP03177(dxs)-   YPK: y1008(dxs)-   YPM: YP_(—)0754(dxs)-   YPA: YPA 2671-   YPN: YPN_(—)0911-   YPP: YPDSF_(—)2812-   YPS: YPTB0939(dxs)-   YPI: YpsIP31758_(—)3112(dxs)-   SFL: SF0357(dxs)-   SFX: S0365(dxs)-   SFV: SFV_(—)0385(dxs)-   SSN: SSON_(—)0397(dxs)-   SBO: SBO_(—)0314(dxs)-   SDY: SDY_(—)0310(dxs)-   ECA: ECA1131(dxs)-   PLU: plu3887(dxs)-   BUC: BU464(dxs)-   BAS: BUsg448(dxs)-   WBR: WGLp144(dxs)-   SGL: SG0656-   KPN: KPN_(—)00372(dxs)-   BFL: Bfl238(dxs)-   BPN: BPEN_(—)244(dxs)-   HIN: HI1439(dxs)-   HIT: NTHI1691(dxs)-   HIP: CGSHiEE_(—)04795-   HIQ: CGSHiGG_(—)01080-   HDU: HD0441(dxs)-   HSO: HS_(—)0905(dxs)-   PMU: PM0532(dxs)-   MSU: MS1059(dxs)-   APL: APL_(—)0207(dxs)-   XFA: XF2249-   XFT: PD1293(dxs)-   XCC: XCC2434(dxs)-   XCB: XC_(—)1678-   XCV: XCV2764(dxs)-   XAC: XAC2565(dxs)-   XOO: XOO2017(dxs)-   XOM: XOO_(—)1900(XOO1900)-   VCH: VC0889-   VVU: VV1_(—)0315-   VVY: VV0868-   VPA: VP0686-   VFI: VF0711-   PPR: PBPRA0805-   PAE: PA4044(dxs)-   PAU: PA14_(—)11550(dxs)-   PAP: PSPA7_(—)1057(dxs)-   PPU: PP_(—)0527(dxs)-   PST: PSPTO_(—)0698(dxs)-   PSB: Psyr_(—)0604-   PSP: PSPPH_(—)0599(dxs)-   PFL: PFL_(—)5510(dxs)-   PFO: Pfl_(—)5007-   PEN: PSEEN0600(dxs)-   PMY: Pmen_(—)3844-   PAR: Psyc_(—)0221(dxs)-   PCR: Pcryo_(—)0245-   ACI: ACIAD3247(dxs)-   SON: SO_(—)1525(dxs)-   SDN: Sden_(—)2571-   SFR: Sfri_(—)2790-   SAZ: Sama_(—)2436-   SBL: Sbal_(—)1357-   SLO: Shew_(—)2771-   SHE: Shewmr4_(—)2731-   SHM: Shewmr7_(—)2804-   SHN: Shewana3_(—)2901-   SHW: Sputw3181_(—)2831-   ILO: IL2138(dxs)-   CPS: CPS_(—)1088(dxs)-   PHA: PSHAa2366(dxs)-   PAT: Patl_(—)1319-   SDE: Sde_(—)3381-   PIN: Ping_(—)2240-   MAQ: Maqu_(—)2438-   MCA: MCA0817(dxs)-   FTU: FTT1018c(dxs)-   FTF: FTF1018c(dxs)-   FTW: FTW_(—)0925(dxs)-   FTL: FTL_(—)1072-   FTH: FTH_(—)1047(dxs)-   FTA: FTA_(—)1131(dxs)-   FTN: FTN_(—)0896(dxs)-   NOC: Noc_(—)1743-   AEH: Mlg_(—)1381-   HCH: HCH_(—)05866(dxs)-   CSA: Csal_(—)0099-   ABO: ABO_(—)2166(dxs)-   AHA: AHA_(—)3321(dxs)-   BCI: BCl_(—)0275(dxs)-   RMA: Rmag_(—)0386-   VOK: COSY_(—)0360(dxs)-   NME: NMB1867-   NMA: NMA0589(dxs)-   NMC: NMC0352(dxs)-   NGO: NG00036-   CVI: CV_(—)2692(dxs)-   RSO: RSc2221(dxs)-   REU: Reut_A0882-   REH: H16_A2732(dxs)-   RME: Rmet_(—)2615-   BMA: BMAA0330(dxs)-   BMV: BMASAVP1_(—)1512(dxs)-   BML: BMA10299_(—)1706(dxs)-   BMN: BMA10247_A0364(dxs)-   BXE: Bxe_B2827-   BUR: Bcep18194_B2211-   BCN: Bcen_(—)4486-   BCH: Bcen2424_(—)3879-   BAM: Bamb_(—)3250-   BPS: BPSS1762(dxs)-   BPM: BURPS1710b_A0842(dxs)-   BPL: BURPS1106A_A2392(dxs)-   BPD: BURPS668_A2534(dxs)-   BTE: BTH_II0614(dxs)-   BPE: BP2798(dxs)-   BPA: BPP2464(dxs)-   BBR: BB1912(dxs)-   RFR: Rfer_(—)2875-   POL: Bpro_(—)1747-   PNA: Pnap_(—)1501-   AJS: Ajs_(—)1038-   MPT: Mpe_A2631-   HAR: HEAR0279(dxs)-   MMS: mma_(—)0331-   NEU: NE1161(dxs)-   NET: Neut_(—)1501-   NMU: Nmul_A0236-   EBA: ebA4439(dxs)-   AZO: azo1198(dxs)-   DAR: Daro_(—)3061-   TBD: Tbd_(—)0879-   MFA: Mfla_(—)2133-   HPY: HP0354(dxs)-   HPJ: jhp0328(dxs)-   HPA: HPAG1_(—)0349-   HHE: HH0608(dxs)-   HAC: Hac_(—)0968(dxs)-   WSU: WS1996-   TDN: Tmden_(—)0475-   CJE: Cj0321(dxs)-   CJR: CJE0366(dxs)-   CJJ: CJJ81176_(—)0343(dxs)-   CJU: C8J_(—)0298(dxs)-   CJD: JJD26997_(—)1642(dxs)-   CFF: CFF8240_(—)0264(dxs)-   CCV: CCV52592_(—)1671(dxs) CCV52592_(—)1722-   CHA: CHAB381_(—)1297(dxs)-   CCO: CCC13826_(—)1594(dxs)-   ABU: Abu_(—)2139(dxs)-   NIS: NIS_(—)0391(dxs)-   SUN: SUN_(—)2055(dxs)-   GSU: GSU0686(dxs-1) GSU1764(dxs-2)-   GME: Gmet_(—)1934 Gmet_(—)2822-   PCA: Pcar_(—)1667-   PPD: Ppro_(—)1191 Ppro_(—)2403-   DVU: DVU1350(dxs)-   DVL: Dvul_(—)1718-   DDE: Dde_(—)2200-   LIP: LI0408(dsx)-   DPS: DP2700-   ADE: Adeh_(—)1097-   MXA: MXAN_(—)4643(dxs)-   SAT: SYN_(—)02456-   SFU: Sfum_(—)1418-   PUB: SAR11_(—)0611(dxs)-   MLO: mlr7474-   MES: Meso_(—)0735-   SME: SMc00972(dxs)-   ATU: Atu0745(dxs)-   ATC: AGR_C_(—)1351-   RET: RHE_CH00913(dxs)-   RLE: RL0973(dxs)-   BME: BMEI1498-   BMF: BAB1_(—)0462(dxs)-   BMS: BR0436(dxs)-   BMB: BruAb1_(—)0458(dxs)-   BOV: BOV_(—)0443(dxs)-   BJA: bl12651(dxs)-   BRA: BRADO2161(dxs)-   BBT: BBta_(—)2479(dxs)-   RPA: RPA0952(dxs)-   RPB: RPB_(—)4460-   RPC: RPC_(—)1149-   RPD: RPD_(—)4305-   RPE: RPE_(—)1067-   NWI: Nwi_(—)0633-   NHA: Nham_(—)0778-   BHE: BH04350(dxs)-   BQU: BQ03540(dxs)-   BBK: BARBAKC583_(—)0400(dxs)-   CCR: CC_(—)2068-   SIL: SPO0247(dxs)-   SIT: TM1040_(—)2920-   RSP: RSP_(—)0254(dxsA) RSP_(—)1134(dxs)-   JAN: Jann_(—)0088 Jann_(—)0170-   RDE: RD1_(—)0101(dxs) RD1_(—)0548(dxs)-   MMR: Mmar10_(—)0849-   HNE: HNE_(—)1838(dxs)-   ZMO: ZMO1234(dxs) ZMO1598(dxs)-   NAR: Saro_(—)0161-   SAL: Sala_(—)2354-   ELI: ELI_(—)12520-   GOX: GOX0252-   GBE: GbCGDNIH1_(—)0221 GbCGDNIH1_(—)2404-   RRU: Rru_A0054 Rru_A2619-   MAG: amb2904-   MGM: Mmc1_(—)1048-   SUS: Acid_(—)1783-   BSU: BG11715(dxs)-   BHA: BH2779-   BAN: BA4400(dxs)-   BAR: GBAA4400(dxs)-   BAA: BA_(—)4853-   BAT: BAS4081-   BCE: BC4176(dxs)-   BCA: BCE_(—)4249(dxs)-   BCZ: BCZK3930(dxs)-   BTK: BT9727_(—)3919(dxs)-   BTL: BALH_(—)3785(dxs)-   BLI: BL01523(dxs)-   BLD: BLi02598(dxs)-   BCL: ABC2462(dxs)-   BAY: RBAM_(—)022600-   BPU: BPUM_(—)2159-   GKA: GK2392-   GTN: GTNG_(—)2322-   LMO: lmo1365(tktB)-   LMF: LMOf2365_(—)1382(dxs)-   LIN: lin1402(tktB)-   LWE: lwe1380(tktB)-   LLA: L108911(dxsA) L123365(dxsB)-   LLC: LACR_(—)1572 LACR_(—)1843-   LLM: llmg_(—)0749(dxsB)-   SAK: SAK_(—)0263-   LPL: lp_(—)2610(dxs)-   LJO: LJ0406-   LAC: LBA0356-   LSL: LSL_(—)0209(dxs)-   LGA: LGAS_(—)0350-   STH: STH1842-   CAC: CAC2077 CA_P0106(dxs)-   CPE: CPE1819-   CPF: CPF_(—)2073(dxs)-   CPR: CPR_(—)1787(dxs)-   CTC: CTC01575-   CNO: NT01CX_(—)1983-   CTH: Cthe_(—)0828-   CDF: CD1207(dxs)-   CBO: CB01881(dxs)-   CBA: CLB_(—)1818(dxs)-   CBH: CLC_(—)1825(dxs)-   CBF: CLI_(—)1945(dxs)-   CKL: CKL_(—)1231(dxs)-   CHY: CHY_(—)1985(dxs)-   DSY: DSY2348-   DRM: Dred_(—)1078-   PTH: PTH_(—)1196(dxs)-   SWO: Swol_(—)0582-   CSC: Csac_(—)1853-   TTE: TTE1298(dxs)-   MTA: Moth_(—)1511-   MPE: MYPE730-   MGA: MGA_(—)1268(dxs)-   MTU: Rv2682c(dxs1) Rv3379c(dxs2)-   MTC: MT2756(dxs)-   MBO: Mb2701c(dxs1) Mb3413c(dxs2)-   MLE: ML1038(dxs)-   MPA: MAP2803c(dxs)-   MAV: MAV_(—)3577(dxs)-   MSM: MSMEG_(—)2776(dxs)-   MMC: Mmcs_(—)2208-   CGL: NCgl1827(cgl1902)-   CGB: cg2083(dxs)-   CEF: CE1796-   CDI: DIP1397(dxs)-   CJK: jk1078(dxs)-   NFA: nfa37410(dxs)-   RHA: RHA1_ro06843-   SCO: SCO6013(SC1C3.01) SCO6768(SC6A5.17)-   SMA: SAV1646(dxs1) SAV2244(dxs2)-   TWH: TWT484-   TWS: TW280(Dxs)-   LXX: Lxx10450(dxs)-   CMI: CMM_(—)1660(dxsA)-   AAU: AAur_(—)1790(dxs)-   PAC: PPA1062-   TFU: Tfu_(—)1917-   FRA: Francci3_(—)1326-   FAL: FRAAL2088(dxs)-   ACE: Acel_(—)1393-   SEN: SACE_(—)1815(dxs) SACE_(—)4351-   BLO: BL1132(dxs)-   BAD: BAD_(—)0513(dxs)-   FNU: FN1208 FN1464-   RBA: RB2143(dxs)-   CTR: CT331(dxs)-   CTA: CTA_(—)0359(dxs)-   CMU: TC0608-   CPN: CPn1060(tktB_(—)2)-   CPA: CP0790-   CPJ: CPj1060(tktB_(—)2)-   CPT: CpB1102-   CCA: CCA00304(dxs)-   CAB: CAB301(dxs)-   CFE: CF0699(dxs)-   PCU: pc0619(dxs)-   TPA: TP0824-   TDE: TDE1910(dxs)-   LIL: LA3285(dxs)-   LIC: LIC10863(dxs)-   LBJ: LBJ_(—)0917(dxs)-   LBL: LBL_(—)0932(dxs)-   SYN: sll1945(dxs)-   SYW: SYNW1292(Dxs)-   SYC: syc1087_c(dxs)-   SYF: Synpcc7942_(—)0430-   SYD: Syncc9605_(—)1430-   SYE: Syncc9902_(—)1069-   SYG: sync_(—)1410(dxs)-   SYR: SynRCC307_(—)1390(dxs)-   SYX: SynWH7803_(—)1223(dxs)-   CYA: CYA_(—)1701(dxs)-   CYB: CYB_(—)1983(dxs)-   TEL: tl10623-   GVI: gl10194-   ANA: alr0599-   AVA: Ava_(—)4532-   PMA: Pro0928(dxs)-   PMM: PMM0907(Dxs)-   PMT: PMT0685(dxs)-   PMN: PMN2A_(—)0300-   PMI: PMT9312_(—)0893-   PMB: A9601_(—)09541(dxs)-   PMC: P9515_(—)09901(dxs)-   PMF: P9303_(—)15371(dxs)-   PMG: P9301_(—)09521(dxs)-   PMH: P9215_(—)09851-   PMJ: P9211_(—)08521-   PME: NATL1_(—)09721(dxs)-   TER: Tery_(—)3042-   BTH: BT_(—)1403 BT_(—)4099-   BFR: BF0873 BF4306-   BFS: BF0796(dxs) BF4114-   PGI: PG2217(dxs)-   CHU: CHU_(—)3643(dxs)-   GFO: GFO_(—)3470(dxs)-   FPS: FP0279(dxs)-   CTE: CT0337(dxs)-   CPH: Cpha266_(—)0671-   PVI: Cvib_(—)0498-   PLT: Plut_(—)0450-   DET: DET0745(dxs)-   DEH: cbdb_A720(dxs)-   DRA: DR_(—)1475-   DGE: Dgeo_(—)0994-   TTH: TTC1614-   TTJ: TTHA0006-   AAE: aq_(—)881-   TMA: TM1770-   PMO: Pmob_(—)1001    Exemplary Acetyl-CoA-Acetyltransferase Nucleic Acids and    Polypeptides-   HSA: 38(ACAT1) 39(ACAT2)-   PTR: 451528(ACAT1)-   MCC: 707653(ACAT1) 708750(ACAT2)-   MMU: 110446(Acat1) 110460(Acat2)-   RNO: 25014(Acat1)-   CFA: 484063(ACAT2) 489421(ACAT1)-   GGA: 418968(ACAT1) 421587(RCJMB04_(—)34i5)-   XLA: 379569(MGC69098) 414622(MGC81403) 414639(MGC81256)    444457(MGC83664)-   XTR: 394562(acat2)-   DRE: 30643(acat2)-   SPU: 759502(LOC759502)-   DME: Dmel_CG10932 Dmel_CG9149-   CEL: T02G5.4 T02G5.7 T02G5.8(kat-1)-   ATH: AT5G48230(ACAT2/EMB1276)-   OSA: 4326136 4346520-   CME: CMA042C CME087C-   SCE: YPL028W(ERG10)-   AGO: AGOS_ADR165c-   PIC: PICST_(—)31707(ERG10)-   CAL: CaO19.1591(erg10)-   CGR: CAGLOL12364g-   SPO: SPBC215.09c-   MGR: MGG_(—)01755 MGG_(—)13499-   ANI: AN1409.2-   AFM: AFUA_(—)6G14200 AFUA_(—)8G04000-   AOR: AO090103000012 AO090103000406-   CNE: CNC05280-   UMA: UM03571.1-   DDI: DDB_(—)0231621-   PFA: PF14_(—)0484-   TET: TTHERM_(—)00091590 TTHERM_(—)00277470 TTHERM_(—)00926980-   TCR: 511003.60-   ECO: b2224(atoB)-   ECJ: JW2218(atoB) JW5453(yqeF)-   ECE: Z4164(yqeF)-   ECS: ECs3701-   ECC: c2767(atoB) c3441(yqeF)-   ECI: UTI89_C2506(atoB) UTI89_C3247(yqeF)-   ECP: ECP_(—)2268 ECP_(—)2857-   ECV: APECO1_(—)3662(yqeF) APECO1_(—)4335(atoB) APECO1_(—)43352(atoB)-   ECX: EcHS_A2365-   STY: STY3164(yqeF)-   STT: t2929(yqeF)-   SPT: SPA2886(yqeF)-   SEC: SC2958(yqeF)-   STM: STM3019(yqeF)-   SFL: SF2854(yqeF)-   SFX: S3052(yqeF)-   SFV: SFV_(—)2922(yqeF)-   SSN: SSON_(—)2283(atoB) SSON_(—)3004(yqeF)-   SBO: SBO_(—)2736(yqeF)-   ECA: ECA1282(atoB)-   ENT: Ent638_(—)3299-   SPE: Spro_(—)0592-   HIT: NTHI0932(atoB)-   XCC: XCC1297(atoB)-   XCB: XC_(—)2943-   XCV: XCV1401(thlA)-   XAC: XAC1348(atoB)-   XOO: XOO1881(atoB)-   XOM: XOO_(—)1778(XOO1778)-   VCH: VCA0690-   VCO: VC0395_(—)0630-   VVU: VV2_(—)0494 VV2_(—)0741-   VVY: VVA1043 VVA1210-   VPA: VPA0620 VPA1123 VPA1204-   PPR: PBPRB1112 PBPRB1840-   PAE: PA2001(atoB) PA2553 PA3454 PA3589 PA3925-   PAU: PA14_(—)38630(atoB)-   PPU: PP_(—)2051(atoB) PP_(—)2215(fadAx) PP_(—)3754 PP_(—)4636-   PPF: Pput_(—)2009 Pput_(—)2403 Pput_(—)3523 Pput_(—)4498-   PST: PSPTO_(—)0957(phbA-1) PSPTO_(—)3164(phbA-2)-   PSB: Psyr_(—)0824 Psyr_(—)3031-   PSP: PSPPH_(—)0850(phbA1) PSPPH_(—)2209(phbA2)-   PFL: PFL_(—)1478(atoB-2) PFL_(—)2321 PFL_(—)3066 PFL_(—)4330(atoB-2)    PFL_(—)5283-   PFO: Pfl_(—)1269 Pfl_(—)1739 Pfl_(—)2074 Pfl_(—)2868-   PEN: PSEEN3197 PSEEN3547(fadAx) PSEEN4635(phbA)-   PMY: Pmen_(—)1138 Pmen_(—)2036 Pmen_(—)3597 Pmen_(—)3662    Pmen_(—)3820-   PAR: Psyc_(—)0252 Psyc_(—)1169-   PCR: Pcryo_(—)0278 Pcryo_(—)1236 Pcryo_(—)1260-   PRW: PsycPRwf 2011-   ACI: ACIAD0694 ACIAD1612 ACIAD2516(atoB)-   SON: SO_(—)1677(atoB)-   SDN: Sden_(—)1943-   SFR: Sfri_(—)1338 Sfri_(—)2063-   SAZ: Sama_(—)1375-   SBL: Sbal_(—)1495-   SBM: Shew185_(—)1489-   SBN: Sbal195_(—)1525-   SLO: Shew_(—)1667 Shew_(—)2858-   SPC: Sputcn32_(—)1397-   SSE: Ssed_(—)1473 Ssed_(—)3533-   SPL: Spea_(—)2783-   SHE: Shewmr4_(—)2597-   SHM: Shewmr7_(—)2664-   SHN: Shewana3_(—)2771-   SHW: Sputw3181_(—)2704-   ILO: IL0872-   CPS: CPS_(—)1605 CPS_(—)2626-   PHA: PSHAa0908 PSHAa1454(atoB) PSHAa1586(atoB)-   PAT: Patl_(—)2923-   SDE: Sde_(—)3149-   PIN: Ping_(—)0659 Ping_(—)2401-   MAQ: Maqu_(—)2117 Maqu_(—)2489 Maqu_(—)2696 Maqu_(—)3162-   CBU: CBU_(—)0974-   LPN: lpg1825(atoB)-   LPF: lpl1789-   LPP: lpp1788-   NOC: Noc_(—)1891-   AEH: Mlg_(—)0688 Mlg_(—)2706-   HHA: Hhal_(—)1685-   HCH: HCH_(—)05299-   CSA: Csal_(—)0301 Csal_(—)3068-   ABO: ABO_(—)0648(fadAx)-   MMW: Mmwyl1_(—)0073 Mmwyl1_(—)3021 Mmwyl1_(—)3053 Mmwyl1_(—)3097    Mmwyl1_(—)4182-   AHA: AHA_(—)2143(atoB)-   CVI: CV_(—)2088(atoB) CV_(—)2790(phaA)-   RSO: RSc0276(atoB) RSc1632(phbA) RSc1637(bktB) RSc1761(RS02948)-   REU: Reut_A0138 Reut_A1348 Reut_A1353 Reut_B4561 Reut_B4738    Reut_B5587 Reut_C5943 Reut_C6062-   REH: H16_A0170 H16_A0867 H16_A0868 H16_A0872 H16_A1297    H16_A1438(phaA) H16_A1445(bktB) H16_A1528 H16_A1713 H16_A1720    H16_A1887 H16_A2148 H16_B0380 H16_B0381 H16_B0406 H16_B0662    H16_B0668 H16_B0759 H16_B1369 H16_B1771-   RME: Rmet_(—)0106 Rmet_(—)1357 Rmet_(—)1362 Rmet_(—)5156-   BMA: BMA1316 BMA1321(phbA) BMA1436-   BMV: BMASAVP1_A1805(bktB) BMASAVP1_A1810(phbA)-   BML: BMA10299_A0086(phbA) BMA10299_A0091-   BMN: BMA10247_(—)1076(bktB) BMA10247_(—)1081(phbA)-   BXE: Bxe_A2273 Bxe_A2335 Bxe_A2342 Bxe_A4255 Bxe_B0377 Bxe_B0739    Bxe_C0332 Bxe_C0574 Bxe_C0915-   BVI: Bcep1808_(—)0519 Bcep1808_(—)1717 Bcep1808_(—)2877    Bcep1808_(—)3594 Bcep1808_(—)4015 Bcep1808_(—)5507 Bcep1808_(—)5644-   BUR: Bcep18194_A3629 Bcep18194_A5080 Bcep18194_A5091 Bcep18194_A6102    Bcep18194_B0263 Bcep18194_B1439 Bcep18194_C6652 Bcep18194_C6802    Bcep18194_C6874 Bcep18194_C7118 Bcep18194_C7151 Bcep18194_C7332-   BCN: Bcen_(—)1553 Bcen_(—)1599 Bcen_(—)2158 Bcen_(—)2563    Bcen_(—)2998 Bcen_(—)6289-   BCH: Bcen2424_(—)0542 Bcen2424_(—)1790 Bcen2424_(—)2772    Bcen2424_(—)5368 Bcen2424_(—)6232 Bcen2424_(—)6276-   BAM: Bamb_(—)0447 Bamb_(—)1728 Bamb_(—)2824 Bamb_(—)4717    Bamb_(—)5771 Bamb_(—)5969-   BPS: BPSL1426 BPSL1535(phbA) BPSL1540-   BPM: BURPS1710b_(—)2325(bktB) BURPS1710b_(—)2330(phbA)    BURPS1710b_(—)2453(atoB-2)-   BPL: BURPS1106A_(—)2197(bktB) BURPS1106A_(—)2202(phbA)-   BPD: BURPS668_(—)2160(bktB) BURPS668_(—)2165(phbA)-   BTE: BTH_(—)12144 BTH_(—)12256 BTH_(—)12261-   PNU: Pnuc_(—)0927-   BPE: BP0447 BP0668 BP2059-   BPA: BPP0608 BPP1744 BPP3805 BPP4216 BPP4361-   BBR: BB0614 BB3364 BB4250 BB4804 BB4947-   RFR: Rfer_(—)0272 Rfer_(—)1000 Rfer_(—)1871 Rfer_(—)2273    Rfer_(—)2561 Rfer_(—)2594 Rfer_(—)3839-   POL: Bpro_(—)1577 Bpro_(—)2140 Bpro_(—)3113 Bpro_(—)4187-   PNA: Pnap_(—)0060 Pnap_(—)0458 Pnap_(—)0867 Pnap_(—)1159    Pnap_(—)2136 Pnap_(—)2804-   AAV: Aave_(—)0031 Aave_(—)2478 Aave_(—)3944 Aave_(—)4368-   AJS: Ajs_(—)0014 Ajs_(—)0124 Ajs_(—)1931 Ajs_(—)2073 Ajs_(—)2317    Ajs_(—)3548 Ajs_(—)3738 Ajs_(—)3776-   VEI: Veis_(—)1331 Veis_(—)3818 Veis_(—)4193-   DAC: Daci_(—)0025 Daci_(—)0192 Daci_(—)3601 Daci_(—)5988-   MPT: Mpe_A1536 Mpe_A1776 Mpe_A1869 Mpe_A3367-   HAR: HEAR0577(phbA)-   MMS: mma_(—)0555-   NEU: NE2262(bktB)-   NET: Neut_(—)0610-   EBA: ebA5202 p2A409(tioL)-   AZO: azo0464(fadA1) azo0469(fadA2) azo2172(th1A)-   DAR: Daro_(—)0098 Daro_(—)3022-   HPA: HPAG1_(—)0675-   HAC: Hac_(—)0958(atoB)-   GME: Gmet_(—)1719 Gmet_(—)2074 Gmet_(—)2213 Gmet_(—)2268    Gmet_(—)3302-   GUR: Gura_(—)3043-   BBA: Bd0404(atoB) Bd2095-   DOL: Dole_(—)0671 Dole_(—)1778 Dole_(—)2160 Dole_(—)2187-   ADE: Adeh_(—)0062 Adeh_(—)2365-   AFW: Anae109_(—)0064 Anae109_(—)1504-   MXA: MXAN_(—)3791-   SAT: SYN_(—)02642-   SFU: Sfum_(—)2280 Sfum_(—)3582-   RPR: RP737-   RCO: RC1134 RC1135-   RFE: RF_(—)0163(paaJ)-   RBE: RBE_(—)0139(paaJ)-   RAK: A1C 05820-   RBO: A1I_(—)07215-   RCM: A1E_(—)04760-   PUB: SAR11_(—)0428(thlA)-   MLO: mlr3847-   MES: Meso_(—)3374-   PLA: Plav_(—)1573 Plav_(—)2783-   SME: SMa1450 SMc03879(phbA)-   SMD: Smed_(—)0499 Smed_(—)3117 Smed_(—)5094 Smed_(—)5096-   ATU: Atu2769(atoB) Atu3475-   ATC: AGR_C_(—)5022(phbA) AGR_L_(—)2713-   RET: RHE_CH04018(phbAch) RHE_PC00068(ypc00040) RHE_PF00014(phbAf)-   RLE: RL4621(phaA) pRL100301 pRL120369-   BME: BMEI0274 BMEII0817-   BMF: BAB1_(—)1783(phbA-1) BAB2_(—)0790(phbA-2)-   BMS: BR1772(phbA-1) BRA0448(phbA-2)-   BMB: BruAb1_(—)1756(phbA-1) BruAb2_(—)0774(phbA-2)-   BOV: BOV_(—)1707(phbA-1)-   OAN: Oant_(—)1130 Oant_(—)3107 Oant_(—)3718 Oant_(—)4020-   BJA: bll0226(atoB) bll3949 bll7400 bll7819 blr3724(phbA)-   BRA: BRADO0562(phbA) BRADO0983(pimB) BRADO3110 BRADO3134(atoB)-   BBT: BBta_(—)3558 BBta_(—)3575(atoB) BBta_(—)5147(pimB)    BBta_(—)7072(pimB)-   BBta_(—)7614(phbA)-   RPA: RPA0513(pcaF) RPA0531 RPA3715(pimB)-   RPB: RPB_(—)0509 RPB_(—)0525 RPB_(—)1748-   RPC: RPC_(—)0504 RPC_(—)0636 RPC_(—)0641 RPC_(—)0832 RPC_(—)1050    RPC_(—)2005 RPC_(—)2194 RPC_(—)2228-   RPD: RPD_(—)0306 RPD_(—)0320 RPD 3105 RPD_(—)3306-   RPE: RPE 0168 RPE_(—)0248 RPE_(—)3827-   NWI: Nwi_(—)3060-   XAU: Xaut_(—)3108 Xaut_(—)4665-   CCR: CC 0510 CC_(—)0894 CC_(—)3462-   SIL: SPO0142(bktB) SPO0326(phbA) SPO0773 SPO3408-   SIT: TM1040_(—)0067 TM1040_(—)2790 TM1040_(—)3026 TM1040_(—)3735-   RSP: RSP_(—)0745 RSP_(—)1354 RSP 3184-   RSH: Rsph17029_(—)0022 Rsph17029_(—)2401 Rsph17029_(—)3179    Rsph17029_(—)3921-   RSQ: Rsph17025_(—)0012 Rsph17025_(—)2466 Rsph17025_(—)2833-   JAN: Jann_(—)0262 Jann_(—)0493 Jann_(—)4050-   RDE: RD1_(—)0025 RD1_(—)0201(bktB) RD1_(—)3394(phbA)-   PDE: Pden_(—)2026 Pden_(—)2663 Pden_(—)2870 Pden_(—)2907    Pden_(—)4811 Pden_(—)5022-   DSH: Dshi_(—)0074 Dshi_(—)3066 Dshi_(—)3331-   MMR: Mmar10_(—)0697-   HNE: HNE_(—)2706 HNE_(—)3065 HNE 3133-   NAR: Saro_(—)0809 Saro_(—)1069 Saro_(—)1222 Saro_(—)2306    Saro_(—)2349-   SAL: Sala_(—)0781 Sala_(—)1244 Sala_(—)2896 Sala 3158-   SWI: Swit_(—)0632 Swit_(—)0752 Swit_(—)2893 Swit_(—)3602    Swit_(—)4887 Swit_(—)5019 Swit_(—)5309-   ELI: ELI_(—)01475 ELI_(—)06705 ELI_(—)12035-   GBE: GbCGDNIH1_(—)0447-   ACR: Acry_(—)1847 Acry_(—)2256-   RRU: Rru_A0274 Rru_A1380 Rru_A1469 Rru_A1946 Rru_A3387-   MAG: amb0842-   MGM: Mmc1_(—)1165-   ABA: Acid345_(—)3239-   BSU: BG11319(mmgA) BG13063(yhfS)-   BHA: BH1997 BH2029 BH3801(mmgA)-   BAN: BA3687 BA4240 BA5589-   BAR: GBAA3687 GBAA4240 GBAA5589-   BAA: BA_(—)0445 BA_(—)4172 BA_(—)4700-   BAT: BAS3418 BAS3932 BAS5193-   BCE: BC3627 BC4023 BC5344-   BCA: BCE_(—)3646 BCE_(—)4076 BCE_(—)5475-   BCZ: BCZK3329(mmgA) BCZK3780(thl) BCZK5044(atoB)-   BCY: Bcer98_(—)2722 Bcer98_(—)3865-   BTK: BT9727_(—)3379(mmgA) BT9727_(—)3765(thl) BT9727_(—)5028(atoB)-   BTL: BALH_(—)3262(mmgA) BALH_(—)3642(fadA) BALH_(—)4843(atoB)-   BLI: BL03925(mmgA)-   BLD: BLi03968(mmgA)-   BCL: ABC0345 ABC2989 ABC3617 ABC3891(mmgA)-   BAY: RBAM_(—)022450-   BPU: BPUM_(—)2374(yhfS) BPUM_(—)2941 BPUM_(—)3373-   OIH: OB06760B06890B26320B3013-   GKA: GK1658 GK3397-   SAU: SA0342 SA0534(vraB)-   SAV: SAV0354 SAV0576(vraB)-   SAM: MW0330 MW0531(vraB)-   SAR: SAR0351(thl) SAR0581-   SAS: SAS0330 SAS0534-   SAC: SACOL0426 SACOL0622(atoB)-   SAB: SAB0304(thl) SAB0526-   SAA: SAUSA300_(—)0355 SAUSA300_(—)0560(vraB)-   SAO: SAOUHSC_(—)00336 SAOUHSC_(—)00558-   SAJ: SaurJH9_(—)0402-   SAH: SaurJH1_(—)0412-   SEP: SE0346 SE2384-   SER: SERP0032 SERP0220-   SHA: SH0510(mvaC) SH2417-   SSP: SSP0325 SSP2145-   LMO: lmo1414-   LMF: LMOf2365_(—)1433-   LIN: lin1453-   LWE: lwe1431-   LLA: L11745(thiL) L25946(fadA)-   LLC: LACR_(—)1665 LACR_(—)1956-   LLM: llmg_(—)0930(thiL)-   SPY: SPy_(—)0140 SPy_(—)1637(atoB)-   SPZ: M5005_Spy_(—)0119 M5005_Spy_(—)0432 M5005_Spy_(—)1344(atoB)-   SPM: spyM18_(—)0136 spyM18_(—)1645(atoB)-   SPG: SpyM3_(—)0108 SpyM3_(—)1378(atoB)-   SPS: SPs0110 SPs0484-   SPH: MGAS10270_Spy0121 MGAS10270_Spy0433 MGAS10270_Spy1461(atoB)-   SPI: MGAS10750_Spy0124 MGAS10750_Spy0452 MGAS10750_Spy1453(atoB)-   SPJ: MGAS2096_Spy0123 MGAS2096_Spy0451 MGAS2096_Spy1365(atoB)-   SPK: MGAS9429_Spy0121 MGAS9429_Spy0431 MGAS9429_Spy1339(atoB)-   SPF: SpyM50447(atoB2)-   SPA: M6_Spy0166 M6_Spy0466 M6_Spy1390-   SPB: M28_Spy0117 M28_Spy0420 M28_Spy1385(atoB)-   SAK: SAK_(—)0568-   LJO: LJ1609-   LAC: LBA0626(thiL)-   LSA: LSA1486-   LDB: Ldb0879-   LBU: LBUL_(—)0804-   LBR: LVIS_(—)2218-   LCA: LSEI_(—)1787-   LGA: LGAS_(—)1374-   LRE: Lreu_(—)0052-   EFA: EF1364-   OOE: OEOE_(—)0529-   STH: STH2913 STH725 STH804-   CAC: CAC2873 CA_P0078(thiL)-   CPE: CPE2195(atoB)-   CPF: CPF_(—)2460-   CPR: CPR_(—)2170-   CTC: CTC00312-   CNO: NTO1CX_(—)0538 NTO1CX_(—)0603-   CDF: CD1059(thlA1) CD2676(thlA2)-   CBO: CB03200(thl)-   CBE: Cbei_(—)0411 Cbei_(—)3630-   CKL: CKL_(—)3696(thlA1) CKL_(—)3697(thlA2) CKL_(—)3698(thlA3)-   AMT: Amet_(—)4630-   AOE: Clos_(—)0084 Clos_(—)0258-   CHY: CHY_(—)1288 CHY_(—)1355(atoB) CHY_(—)1604 CHY_(—)1738-   DSY: DSY0632 DSY0639 DSY1567 DSY1710 DSY2402 DSY3302-   DRM: Dred_(—)0400 Dred_(—)1491 Dred_(—)1784 Dred_(—)1892-   SWO: Swol_(—)0308 Swol_(—)0675 Swol_(—)0789 Swol_(—)1486    Swol_(—)1934 Swol_(—)2051-   TTE: TTE0549(paaJ)-   MTA: Moth_(—)1260 MTU: Rv1135A Rv1323(fadA4) Rv3546(fadA5)-   MTC: MT1365(phbA)-   MBO: Mb1167 Mb1358(fadA4) Mb3576(fadA5) Mb3586c(fadA6)-   MBB: BCG_(—)1197 BCG_(—)1385(fadA4) BCG_(—)3610(fadA5)    BCG_(—)3620c(fadA6)-   MLE: ML1158(fadA4)-   MPA: MAP2407c(fadA3) MAP2436c(fadA4)-   MAV: MAV_(—)1544 MAV_(—)1573 MAV_(—)1863 MAV_(—)5081-   MSM: MSMEG_(—)2224 MSMEG_(—)4920-   MUL: MUL_(—)0357-   MVA: Mvan_(—)1976 Mvan_(—)1988 Mvan_(—)4305 Mvan_(—)4677    Mvan_(—)4891-   MGI: Mflv_(—)1347 Mflv_(—)1484 Mflv_(—)2040 Mflv_(—)2340    Mflv_(—)4356 Mflv_(—)4368-   MMC: Mmcs_(—)1758 Mmcs_(—)1769 Mmcs_(—)3796 Mmcs_(—)3864-   MKM: Mkms_(—)0251 Mkms_(—)1540 Mkms_(—)1805 Mkms_(—)1816    Mkms_(—)2836 Mkms_(—)3159-   Mkms_(—)3286 Mkms_(—)3869 Mkms_(—)3938 Mkms_(—)4227 Mkms_(—)4411    Mkms_(—)4580 Mkms_(—)4724 Mkms_(—)4764 Mkms_(—)4776-   MJL: Mjls_(—)0231 Mjls_(—)1739 Mjls_(—)1750 Mjls_(—)2819    Mjls_(—)3119 Mjls_(—)3235 Mjls_(—)3800 Mjls_(—)3850 Mjls_(—)4110    Mjls_(—)4383 Mjls_(—)4705 Mjls_(—)4876 Mjls_(—)5018 Mjls_(—)5063    Mjls_(—)5075-   CGL: NCg12309(cg12392)-   CGB: cg2625(pcaF)-   CEF: CE0731 CE2295-   CJK: jk1543(fadA3)-   NFA: nfal0750(fadA4)-   RHA: RHA1_ro01455 RHA1_ro01623 RHA1_ro01876 RHA1_ro02517(catF)    RHA1_ro03022 RHA1_ro03024 RHA1_ro03391 RHA1_ro03892 RHA1_ro04599    RHA1_ro05257 RHA1_ro08871-   SCO: SCO5399(SC8F4.03)-   SMA: SAV1384(fadA5) SAV2856(fadA1)-   ART: Arth_(—)1160 Arth_(—)2986 Arth_(—)3268 Arth_(—)4073-   NCA: Noca_(—)1371 Noca_(—)1797 Noca_(—)1828 Noca_(—)2764    Noca_(—)4142-   TFU: Tfu_(—)1520 Tfu_(—)2394-   FRA: Francci3_(—)3687-   FRE: Franean1_(—)1044 Franean1_(—)2711 Franean1_(—)2726    Franean1_(—)3929 Franean1_(—)4037 Franean1_(—)4577-   FAL: FRAAL2514 FRAAL2618 FRAAL5910(atoB)-   ACE: Acel_(—)0626 Acel_(—)0672-   SEN: SACE_(—)1192(mmgA) SACE_(—)2736(fadA6) SACE_(—)4011(catF)    SACE_(—)6236(fadA4)-   STP: Strop_(—)3610-   SAQ: Sare_(—)1316 Sare_(—)3991-   RXY: Rxyl_(—)1582 Rxyl_(—)1842 Rxyl_(—)2389 Rxyl_(—)2530-   FNU: FN0495-   BGA: BG0110(fadA)-   BAF: BAPKO_(—)0110(fadA)-   LIL: LA0457(thiL1) LA0828(thiL2) LA4139(fadA)-   LIC: LIC10396(phbA)-   LBJ: LBJ_(—)2862(paaJ-4)-   LBL: LBL_(—)0209(paaJ-4)-   SYN: s1r1993(phaA)-   SRU: SRU_(—)1211(atoB) SRU_(—)1547-   CHU: CHU_(—)1910(atoB)-   GFO: GFO_(—)1507(atoB)-   FJO: Fjoh_(—)4612-   FPS: FP0770 FP1586 FP1725-   RRS: RoseRS_(—)3911 RoseRS_(—)4348-   RCA: Rcas_(—)0702 Rcas_(—)3206-   HAU: Haur_(—)0522-   DRA: DR_(—)1072 DR_(—)1428 DR_(—)1960 DR_(—)2480 DR_A0053-   DGE: Dgeo_(—)0755 Dgeo_(—)1305 Dgeo_(—)1441 Dgeo_(—)1883-   TTH: TTC0191 TTC0330-   TTJ: TTHA0559-   TME: Tmel_(—)1134-   FNO: Fnod_(—)0314-   PMO: Pmob_(—)0515-   HMA: rrnAC0896(acaB3) rrnAC2815(aca2) rrnAC3497(yqeF) rrnB0240(aca1)    rrnB0242(acaB2) rrnB0309(acaB1)-   TAC: Ta0582-   TVO: TVN0649-   PTO: PT01505-   APE: APE_(—)2108-   SSO: SSO2377(acaB-4)-   STO: ST0514-   SAI: Saci_(—)0963 Saci_(—)1361(acaB1)-   MSE: Msed_(—)0656-   PAI: PAE1220-   PIS: Pisl_(—)0029 Pisl_(—)1301-   PCL: Pcal_(—)0781-   PAS: Pars_(—)0309 Pars_(—)1071-   CMA: Cmaq_(—)1941    Exemplary HMG-CoA Synthase Nucleic Acids and Polypeptides-   HSA: 3157(HMGCS1) 3158(HMGCS2)-   PTR: 457169(HMGCS2) 461892(HMGCS1)-   MCC: 702553(HMGCS1) 713541(HMGCS2)-   MMU: 15360(Hmgcs2) 208715(Hmgcs1)-   RNO: 24450(Hmgcs2) 29637(Hmgcs1)-   CFA: 479344(HMGCS1) 607923(HMGCS2)-   BTA: 407767(HMGCS1)-   SSC: 397673(CH242-38B5.1)-   GGA: 396379(HMGCS1)-   XLA: 380091(hmgcs1) 447204(MGC80816)-   DRE: 394060(hmgcs1)-   SPU: 578259(L00578259)-   DME: Dmel_CG4311(Hmgs)-   CEL: F25B4.6-   ATH: AT4G11820(BAP1)-   OSA: 4331418 4347614-   CME: CMM189C-   SCE: YML126C(ERG13)-   AGO: AGOS_ADL356C-   PIC: PICST_(—)83020-   CAL: CaO19_(—)7312(CaO19.7312)-   CGR: CAGL0H04081g-   SPO: SPAC4F8.14c(hcs)-   MGR: MGG_(—)01026-   ANI: AN4923.2-   AFM: AFUA_(—)3G10660 AFUA_(—)8G07210-   AOR: AO090003000611 AO090010000487-   CNE: CNC05080 CNG02670-   UMA: UM05362.1-   ECU: ECU10_(—)0510-   DDI: DDBDRAFT_(—)0217522 DDB_(—)0219924(hgsA)-   TET: TTHERM_(—)00691190-   TBR: Tb927.8.6110-   YPE: YP01457-   YPK: y2712(pksG)-   YPM: YP_(—)1349(pksG)-   YPA: YPA_(—)0750-   YPN: YPN_(—)2521-   YPP: YPDSF_(—)1517-   YPS: YPTB1475-   CBD: COXBU7E912_(—)1931-   TCX: Tcr_(—)1719-   DNO: DNO 0799-   BMA: BMAAl212-   BPS: BPSS1002-   BPM: BURPS1710b_A2613-   BPL: BURPS1106A_A1384-   BPD: BURPS668_A1470-   BTE: BTH_II1670-   MXA: MXAN_(—)3948(tac) MXAN_(—)4267(mvaS)-   BSU: BG10926(pksG)-   OIH: OB2248-   SAU: SA2334(mvaS)-   SAV: SAV2546(mvaS)-   SAM: MW2467(mvaS)-   SAR: SAR2626(mvaS)-   SAS: SAS2432-   SAC: SACOL2561-   SAB: SAB2420(mvaS)-   SAA: SAUSA300_(—)2484-   SAO: SAOUHSC_(—)02860-   SAJ: SaurJH9_(—)2569-   SAH: SaurJH1_(—)2622-   SEP: SE2110-   SER: SERP2122-   SHA: SH0508(mvaS)-   SSP: SSP0324-   LMO: lmo1415-   LMF: LMOf2365_(—)1434(mvaS)-   LIN: lin1454-   LWE: lwe1432(mvaS)-   LLA: L13187(hmcM)-   LLC: LACR_(—)1666-   LLM: llmg_(—)0929(hmcM)-   SPY: SPy_(—)0881(mvaS.2)-   SPZ: M5005—Spy_(—)0687(mvaS.1)-   SPM: spyM18_(—)0942(mvaS2)-   SPG: SpyM3_(—)0600(mvaS.2)-   SPS: SPs1253-   SPH: MGAS10270_Spy0745(mvaS1)-   SPI: MGAS10750_Spy0779(mvaS1)-   SPJ: MGAS2096_Spy0759(mvaS1)-   SPK: MGAS9429_Spy0743(mvaS1)-   SPF: SpyM51121(mvaS)-   SPA: M6_Spy0704-   SPB: M28_Spy0667(mvaS.1)-   SPN: SP_(—)1727-   SPR: spr1571(mvaS)-   SPD: SPD_(—)1537(mvaS)-   SAG: SAG1316-   SAN: gbs1386-   SAK: SAK_(—)1347-   SMU: SMU.943c-   STC: str0577(mvaS)-   STL: stu0577(mvaS)-   STE: STER_(—)0621-   SSA: SSA_(—)0338(mvaS)-   SSU: SSU05_(—)1641-   SSV: SSU98_(—)1652-   SGO: SGO_(—)0244-   LPL: lp_(—)2067(mvaS)-   LJO: LJ1607-   LAC: LBA0628(hmcS)-   LSA: LSA1484(mvaS)-   LSL: LSL_(—)0526-   LDB: Ldb0881(mvaS)-   LBU: LBUL_(—)0806-   LBR: LVIS_(—)1363-   LCA: LSEI_(—)1785-   LGA: LGAS_(—)1372-   LRE: Lreu_(—)0676-   PPE: PEPE_(—)0868-   EFA: EF1363-   OOE: OEOE_(—)0968-   LME: LEUM_(—)1184-   NFA: nfa22120-   SEN: SACE_(—)4570(pksG)-   BBU: BB0683-   BGA: BG0706-   BAF: BAPKO_(—)0727-   FJO: Fjoh_(—)0678-   HAL: VNG1615G(mvaB)-   HMA: rrnAC1740(mvaS)-   HWA: HQ2868A(mvaB)-   NPH: NP2608A(mvaB_(—)1) NP4836A(mvaB_(—)2)    Exemplary Hydroxymethylglutaryl-CoA Reductase Nucleic Acids and    Polypeptides-   HSA: 3156(HMGCR)-   PTR: 471516(HMGCR)-   MCC: 705479(HMGCR)-   MMU: 15357(Hmgcr)-   RNO: 25675(Hmgcr)-   CFA: 479182(HMGCR)-   BTA: 407159(HMGCR)-   GGA: 395145(RCJMB04_(—)14m24)-   SPU: 373355(LOC373355)-   DME: Dmel_CG10367 (Hmgcr)-   CEL: F08F8.2-   OSA: 4347443-   SCE: YLR450W(HMG2) YML075C(HMG1)-   AGO: AGOS_AER152W-   CGR: CAGLOL11506g-   SPO: SPCC162.09c(hmg1)-   ANI: AN3817.2-   AFM: AFUA_(—)1G11230 AFUA_(—)2G03700-   AOR: AO090103000311 AO090120000217-   CNE: CNF04830-   UMA: UM03014.1-   ECU: ECU10_(—)1720-   DDI: DDB_(—)0191125(hmgA) DDB_(—)0215357(hmgB)-   TBR: Tb927.6.4540-   TCR: 506831.40 509167.20-   LMA: LmjF30.3190-   VCH: VCA0723-   VCO: VC0395_(—)0662-   VVU: VV2_(—)0117-   VVY: VVA0625-   VPA: VPA0968-   VFI: VFA0841-   PAT: Patl_(—)0427-   CBU: CBU_(—)0030 CBU_(—)0610-   CBD: COXBU7E912_(—)0151 COXBU7E912_(—)0622(hmgA)-   TCX: Tcr_(—)1717-   DNO: DNO 0797-   CVI: CV_(—)1806-   SUS: Acid_(—)5728 Acid_(—)6132-   SAU: SA2333(mvaA)-   SAV: SAV2545(mvaA)-   SAM: MW2466(mvaA)-   SAB: SAB2419c(mvaA)-   SEP: SE2109-   LWE: lwe0819(mvaA)-   LLA: L10433(mvaA)-   LLC: LACR_(—)1664-   LLM: llmg_(—)0931(mvaA)-   SPY: SPy_(—)0880(mvaS.1)-   SPM: spyM18_(—)0941(mvaS1)-   SPG: SpyM3_(—)0599(mvaS.1)-   SPS: SPs1254-   SPH: MGAS10270_Spy0744-   SPI: MGAS10750_Spy0778-   SPJ: MGAS2096_Spy0758-   SPK: MGAS9429_Spy0742-   SPA: M6_Spy0703-   SPN: SP_(—)1726-   SAG: SAG1317-   SAN: gbs1387-   STC: str0576(mvaA)-   STL: stu0576(mvaA)-   STE: STER_(—)0620-   SSA: SSA_(—)0337(mvaA)-   LPL: lp_(—)0447(mvaA)-   LJO: LJ1608-   LSL: LSL_(—)0224-   LBR: LVIS_(—)0450-   LGA: LGAS_(—)1373-   EFA: EF1364-   NFA: nfa22110-   BGA: BG0708(mvaA)-   SRU: SRU_(—)2422-   FPS: FP2341-   MMP: MMP0087(hmgA)-   MMQ: MmarC5_(—)1589-   MAC: MA3073(hmgA)-   MBA: Mbar_A1972-   MMA: MM_(—)0335-   MBU: Mbur_(—)1098-   MHU: Mhun_(—)3004-   MEM: Memar_(—)2365-   MBN: Mboo_(—)0137-   MTH: MTH562-   MST: Msp_(—)0584(hmgA)-   MSI: Msm_(—)0227-   MKA: MK0355(HMG1)-   AFU: AF1736(mvaA)-   HAL: VNG1875G(mvaA)-   HMA: rrnAC3412(mvaA)-   HWA: HQ3215A(hmgR)-   NPH: NP0368A(mvaA_(—)2) NP2422A(mvaA_(—)1)-   TAC: Ta0406m-   TVO: TVN1168-   PTO: PT01143-   PAB: PAB2106(mvaA)-   PFU: PF1848-   TKO: TK0914-   RCI: RCIX1027(hmgA) RCIX376(hmgA)-   APE: APE_(—)1869-   IHO: Igni_(—)0476-   HBU: Hbut_(—)1531-   SSO: SS00531-   STO: ST1352-   SAI: Saci_(—)1359-   PAI: PAE2182-   PIS: Pisl_(—)0814-   PCL: Pcal_(—)1085-   PAS: Pars_(—)0796    Exemplary Mevalonate Kinase Nucleic Acids and Polypeptides-   HSA: 4598(MVK)-   MCC: 707645(MVK)-   MMU: 17855(Mvk)-   RNO: 81727(Mvk)-   CFA: 486309(MVK)-   BTA: 505792(MVK)-   GGA: 768555(MVK)-   DRE: 492477(zgc:103473)-   SPU: 585785(LOC585785)-   DME: Dmel_CG33671-   OSA: 4348331-   SCE: YMR208W(ERG12)-   AGO: AGOS_AER335W-   PIC: PICST_(—)40742(ERG12)-   CGR: CAGLOF03861g-   SPO: SPAC13G6.11c-   MGR: MGG_(—)06946-   ANI: AN3869.2-   AFM: AFUA_(—)4G07780-   AOR: AO090023000793-   CNE: CNK01740-   ECU: ECU09_(—)1780-   DDI: DDBDRAFT_(—)0168621-   TET: TTHERM_(—)00637680-   TBR: Tb927.4.4070-   TCR: 436521.9 509237.10-   LMA: LmjF31.0560-   CBU: CBU_(—)0608 CBU_(—)0609-   CBD: COXBU7E912_(—)0620(mvk)-   LPN: lpg2039-   LPF: lpl2017-   LPP: lpp 2022-   BBA: Bd1027(1 mbP) Bd1630(mvk)-   MXA: MXAN_(—)5019(mvk)-   OIH: OB0225-   SAU: SA0547(mvaK1)-   SAV: SAV0590(mvaK1)-   SAM: MW0545(mvaK1)-   SAR: SAR0596(mvaK1)-   SAS: SAS0549-   SAC: SACOL0636(mvk)-   SAB: SAB0540(mvaK1)-   SAA: SAUSA300_(—)0572(mvk)-   SAO: SAOUHSC_(—)00577-   SEP: SE0361-   SER: SERP0238(mvk)-   SHA: SH2402(mvaK1)-   SSP: SSP2122-   LMO: lmo0010-   LMF: LMOf2365_(—)0011-   LIN: lin0010-   LWE: lwe0011(mvk)-   LLA: L7866(yeaG)-   LLC: LACR_(—)0454-   LLM: llmg_(—)0425(mvk)-   SPY: SPy_(—)0876(mvaK1)-   SPZ: M5005_Spy_(—)0682(mvaK1)-   SPM: spyM18_(—)0937(mvaK1)-   SPG: SpyM3_(—)0595(mvaK1)-   SPS: SPs1258-   SPH: MGAS10270_Spy0740(mvaK1)-   SPI: MGAS10750_Spy0774(mvaK1)-   SPJ: MGAS2096_Spy0753(mvaK1)-   SPK: MGAS9429_Spy0737(mvaK1)-   SPF: SpyM51126(mvaK1)-   SPA: M6_Spy0699-   SPB: M28_Spy0662(mvaK1)-   SPN: SP_(—)0381-   SPR: spr0338(mvk)-   SPD: SPD_(—)0346(mvk)-   SAG: SAG1326-   SAN: gbs1396-   SAK: SAK_(—)1357(mvk)-   SMU: SMU.181-   STC: str0559(mvaK1)-   STL: stu0559(mvaK1)-   STE: STER_(—)0598-   SSA: SSA_(—)0333(mvaK1)-   SSU: SSU05_(—)0289-   SSV: SSU98_(—)0285-   SGO: SGO_(—)0239(mvk)-   LPL: lp_(—)1735(mvaK1)-   LJO: LJ1205-   LAC: LBA1167(mvaK)-   LSA: LSA0908(mvaK1)-   LSL: LSL_(—)0685(eRG)-   LDB: Ldb0999(mvk)-   LBU: LBUL_(—)0906-   LBR: LVIS_(—)0858-   LCA: LSEI_(—)1491-   LGA: LGAS_(—)1033-   LRE: Lreu_(—)0915-   PPE: PEPE_(—)0927-   EFA: EF0904(mvk)-   OOE: OEOE_(—)1100-   LME: LEUM_(—)1385-   NFA: nfa22070-   BGA: BG0711-   BAF: BAPKO_(—)0732-   FPS: FP0313-   MMP: MMP1335-   MAE: Maeo_(—)0775-   MAC: MA0602(mvk)-   MBA: Mbar_A1421-   MMA: MM_(—)1762-   MBU: Mbur_(—)2395-   MHU: Mhun_(—)2890-   MEM: Memar_(—)1812-   MBN: Mboo_(—)2213-   MST: Msp_(—)0858(mvk)-   MSI: Msm_(—)1439-   MKA: MK0993(ERG12)-   HAL: VNG1145G(mvk)-   HMA: rrnAC0077(mvk)-   HWA: HQ2925A(mvk)-   NPH: NP2850A(mvk)-   PTO: PT01352-   PHO: PH1625-   PAB: PAB0372(mvk)-   PFU: PF1637(mvk)-   TKO: TK1474-   RCI: LRC399(mvk)-   APE: APE_(—)2439-   HBU: Hbut_(—)0877-   SSO: SS00383-   STO: ST2185-   SAI: Saci_(—)2365(mvk)-   MSE: Msed_(—)1602-   PAI: PAE3108-   PIS: Pisl_(—)0467-   PCL: Pcal_(—)1835    Exemplary Phosphomevalonate Kinase Nucleic Acids and Polypeptides-   HSA: 10654(PMVK)-   PTR: 457350(PMVK)-   MCC: 717014(PMVK)-   MMU: 68603(Pmvk)-   CFA: 612251(PMVK)-   BTA: 513533(PMVK)-   DME: Dmel_CG10268-   ATH: AT1G31910-   OSA: 4332275-   SCE: YMR220W(ERG8)-   AGO: AGOS_AER354W-   PIC: PICST_(—)52257(ERG8)-   CGR: CAGLOF03993g-   SPO: SPAC343.01c-   MGR: MGG_(—)05812-   ANI: AN2311.2-   AFM: AFUA_(—)5G10680-   AOR: AO090010000471-   CNE: CNM00100-   UMA: UM00760.1-   DDI: DDBDRAFT_(—)0184512-   TBR: Tb09.160.3690-   TCR: 507913.20 508277.140-   LMA: LmjF15.1460-   MXA: MXAN_(—)5017-   OIH: OB0227-   SAU: SA0549(mvaK2)-   SAV: SAV0592(mvaK2)-   SAM: MW0547(mvaK2)-   SAR: SAR0598(mvaK2)-   SAS: SAS0551-   SAC: SACOL0638-   SAB: SAB0542(mvaK2)-   SAA: SAUSA300_(—)0574-   SAO: SAOUHSC_(—)00579-   SAJ: SaurJH9_(—)0615-   SEP: SE0363-   SER: SERP0240-   SHA: SH2400(mvaK2)-   SSP: SSP2120-   LMO: lmo0012-   LMF: LMOf2365_(—)0013-   LIN: lin0012-   LWE: lwe0013-   LLA: L10014(yebA)-   LLC: LACR_(—)0456-   LLM: llmg_(—)0427-   SPY: SPy_(—)0878(mvaK2)-   SPZ: M5005_Spy_(—)0684(mvaK2)-   SPM: spyM18_(—)0939-   SPG: SpyM3_(—)0597(mvaK2)-   SPS: SPs1256-   SPH: MGAS10270_Spy0742(mvaK2)-   SPI: MGAS10750_Spy0776(mvaK2)-   SPJ: MGAS2096_Spy0755(mvaK2)-   SPK: MGAS9429_Spy0739(mvaK2)-   SPF: SpyM51124(mvaK2)-   SPA: M6_Spy0701-   SPB: M28_Spy0664(mvaK2)-   SPN: SP_(—)0383-   SPR: spr0340(mvaK2)-   SPD: SPD_(—)0348(mvaK2)-   SAG: SAG1324-   SAN: gbs1394-   SAK: SAK_(—)1355-   SMU: SMU.938-   STC: str0561(mvaK2)-   STL: stu0561(mvaK2)-   STE: STER_(—)0600-   SSA: SSA_(—)0335(mvaK2)-   SSU: SSU05_(—)0291-   SSV: SSU98_(—)0287-   SGO: SGO_(—)0241-   LPL: lp_(—)1733(mvaK2)-   LJO: LJ1207-   LAC: LBA1169-   LSA: LSA0906(mvaK2)-   LSL: LSL_(—)0683-   LDB: Ldb0997(mvaK)-   LBU: LBUL_(—)0904-   LBR: LVIS_(—)0860-   LCA: LSEI_(—)1092-   LGA: LGAS_(—)1035-   LRE: Lreu_(—)0913-   PPE: PEPE_(—)0925-   EFA: EF0902-   NFA: nfa22090-   BGA: BG0710-   BAF: BAPKO_(—)0731-   NPH: NP2852A-   SSO: SSO2988-   STO: ST0978-   SAI: Saci_(—)1244    Exemplary Diphosphomevalonate Decarboxylase Nucleic Acids and    Polypeptides-   HSA: 4597(MVD)-   PTR: 468069(MVD)-   MCC: 696865(MVD)-   MMU: 192156(Mvd)-   RNO: 81726(Mvd)-   CFA: 489663(MVD)-   GGA: 425359(MVD)-   DME: Dmel_CG8239-   SCE: YNR043W(MVD1)-   AGO: AGOS_AGL232C-   PIC: PICST_(—)90752-   CGR: CAGL0C03630g-   SPO: SPAC24C9.03-   MGR: MGG_(—)09750-   ANI: AN4414.2-   AFM: AFUA_(—)4G07130-   AOR: AO090023000862-   CNE: CNL04950-   UMA: UM05179.1-   DDI: DDBDRAFT_(—)0218058-   TET: TTHERM_(—)00849200-   TBR: Tb10.05.0010 Tb10.61.2745-   TCR: 507993.330 511281.40-   LMA: LmjF18.0020-   CBU: CBU_(—)0607(mvaD)-   CBD: COXBU7E912_(—)0619(mvaD)-   LPN: lpg2040-   LPF: lpl2018-   LPP: lpp2023-   TCX: Tcr_(—)1734-   DNO: DNO_(—)0504(mvaD)-   BBA: Bd1629-   MXA: MXAN_(—)5018(mvaD)-   OIH: OB0226-   SAU: SA0548(mvaD)-   SAV: SAV0591(mvaD)-   SAM: MW0546(mvaD)-   SAR: SAR0597(mvaD)-   SAS: SAS0550-   SAC: SACOL0637(mvaD)-   SAB: SAB0541(mvaD)-   SAA: SAUSA300_(—)0573(mvaD)-   SAO: SAOUHSC_(—)00578-   SAJ: SaurJH9_(—)0614-   SAH: SaurJH1_(—)0629-   SEP: SE0362-   SER: SERP0239(mvaD)-   SHA: SH2401(mvaD)-   SSP: SSP2121-   LMO: lmo0011-   LMF: LMOf2365_(—)0012(mvaD)-   LIN: lin0011-   LWE: lwe0012(mvaD)-   LLA: L9089(yeaH)-   LLC: LACR_(—)0455-   LLM: llmg_(—)0426(mvaD)-   SPY: SPy_(—)0877(mvaD)-   SPZ: M5005_Spy_(—)0683(mvaD)-   SPM: spyM18_(—)0938(mvd)-   SPG: SpyM3_(—)0596(mvaD)-   SPS: SPs1257-   SPH: MGAS10270_Spy0741(mvaD)-   SPI: MGAS10750_Spy0775(mvaD)-   SPJ: MGAS2096_Spy0754(mvaD)-   SPK: MGAS9429_Spy0738(mvaD)-   SPF: SpyM51125(mvaD)-   SPA: M6_Spy0700-   SPB: M28_Spy0663(mvaD)-   SPN: SP_(—)0382-   SPR: spr0339(mvdl)-   SPD: SPD_(—)0347(mvaD)-   SAG: SAG1325(mvaD)-   SAN: gbs1395-   SAK: SAK_(—)1356(mvaD)-   SMU: SMU.937-   STC: str0560(mvaD)-   STL: stu0560(mvaD)-   STE: STER_(—)0599-   SSA: SSA_(—)0334(mvaD)-   SSU: SSU05_(—)0290-   SSV: SSU98_(—)0286-   SGO: SGO_(—)0240(mvaD)-   LPL: lp_(—)1734(mvaD)-   LJO: LJ1206-   LAC: LBA1168(mvaD)-   LSA: LSA0907(mvaD)-   LSL: LSL_(—)0684-   LDB: Ldb0998(mvaD)-   LBU: LBUL_(—)0905-   LBR: LVIS_(—)0859-   LCA: LSEI_(—)1492-   LGA: LGAS_(—)1034-   LRE: Lreu_(—)0914-   PPE: PEPE_(—)0926-   EFA: EF0903(mvaD)-   LME: LEUM_(—)1386-   NFA: nfa22080-   BBU: BB0686-   BGA: BG0709-   BAF: BAPKO_(—)0730-   GFO: GFO_(—)3632-   FPS: FP0310(mvaD)-   HAU: Haur_(—)1612-   HAL: VNG0593G(dmd)-   HMA: rrnAC1489(dmd)-   HWA: HQ1525A(mvaD)-   NPH: NP1580A(mvaD)-   PTO: PT00478 PT01356-   SSO: SSO2989-   STO: ST0977-   SAI: Saci_(—)1245(mvd)-   MSE: Msed_(—)1576    Exemplary Isopentenyl Phosphate Kinases (IPK) Nucleic Acids and    Polypeptides-   Methanobacterium thermoautotrophicum gi|2621082-   Methanococcus jannaschii DSM 2661 gi|1590842;-   Methanocaldococcus jannaschii gi|1590842-   Methanothermobacter thermautotrophicus gi|2621082-   Picrophilus torridus DSM9790 (IG-57) gi|48477569-   Pyrococcus abyssi gi|14520758-   Pyrococcus horikoshii OT3 gi|3258052-   Archaeoglobus fulgidus DSM4304 gi|2648231    Exemplary Isopentenyl-Diphosphate Delta-Isomerase (IDI) Nucleic    Acids and Polypeptides-   HSA: 3422(IDI1) 91734(ID12)-   PTR: 450262(ID12) 450263(IDI1)-   MCC: 710052(LOC710052) 721730(LOC721730)-   MMU: 319554(Idi1)-   RNO: 89784(Idi1)-   GGA: 420459(IDI1)-   XLA: 494671(LOC494671)-   XTR: 496783(idi2)-   SPU: 586184(L00586184)-   CEL: K06H7.9(idi-1)-   ATH: AT3G02780(IPP2)-   OSA: 4338791 4343523-   CME: CMB062C-   SCE: YPL117C(IDI1)-   AGO: AGOS_ADL268C-   PIC: PICST_(—)68990(IDI1)-   CGR: CAGLOJ06952g-   SPO: SPBC106.15(idi1)-   ANI: AN0579.2-   AFM: AFUA_(—)6G11160-   AOR: AO090023000500-   CNE: CNA02550-   UMA: UM04838.1-   ECU: ECU02_(—)0230-   DDI: DDB_(—)0191342(ipi)-   TET: TTHERM_(—)00237280 TTHERM_(—)00438860-   TBR: Tb09.211.0700-   TCR: 408799.19 510431.10-   LMA: LmjF35.5330-   EHI: 46.t00025-   ECO: b2889(idi)-   ECJ: JW2857(idi)-   ECE: Z4227-   ECS: ECs3761-   ECC: c3467-   ECI: UTI89_C3274-   ECP: ECP_(—)2882-   ECV: APECO1_(—)3638-   ECW: EcE24377A_(—)3215(idi)-   ECX: EcHS_A3048-   STY: STY3195-   STT: t2957-   SPT: SPA2907(idi)-   SEC: SC2979(idi)-   STM: STM3039(idi)-   SFL: SF2875(idi)-   SFX: S3074-   SFV: SFV_(—)2937-   SSN: SSON_(—)3042 SSON_(—)3489(yhfK)-   SBO: SBO_(—)3103-   SDY: SDY_(—)3193-   ECA: ECA2789-   PLU: plu3987-   ENT: Ent638_(—)3307-   SPE: Spro_(—)2201-   VPA: VPA0278-   VFI: VF0403-   PPR: PBPRA0469(mvaD)-   PEN: PSEEN4850-   CBU: CBU_(—)0607(mvaD)-   CBD: COXBU7E912_(—)0619(mvaD)-   LPN: lpg2051-   LPF: lpl2029-   LPP: lpp2034-   TCX: Tcr_(—)1718-   HHA: Hhal_(—)1623-   DNO: DNO_(—)0798-   EBA: ebA5678 p2A143-   DVU: DVU1679(idi)-   DDE: Dde_(—)1991-   LIP: LI1134-   BBA: Bd1626-   AFW: Anae109_(—)4082-   MXA: MXAN_(—)5021(fni)-   RPR: RP452-   RTY: RT0439(idi)-   RCO: RC0744-   RFE: RF_(—)0785(fni)-   RBE: RBE_(—)0731(fni)-   RAK: A1C_(—)04190-   RBO: A1I_(—)04755-   RCM: A1E_(—)02555-   RR1: A1G_(—)04195-   MLO: mlr6371-   RET: RHE_PD00245(ypd00046)-   XAU: Xaut_(—)4134-   SIL: SPO0131-   SIT: TM1040_(—)3442-   RSP: RSP_(—)0276-   RSH: Rsph17029_(—)1919-   RSQ: Rsph17025_(—)1019-   JAN: Jann_(—)0168-   RDE: RD1_(—)0147(idi)-   DSH: Dshi_(—)3527-   BSU: BG11440(ypgA)-   BAN: BA1520-   BAR: GBAA1520-   BAA: BA_(—)2041-   BAT: BAS1409-   BCE: BC1499-   BCA: BCE_(—)1626-   BCZ: BCZK1380(fni)-   BCY: Bcer98_(—)1222-   BTK: BT9727_(—)1381(fni)-   BTL: BALH_(—)1354-   BLI: BL02217(fni)-   BLD: BLi02426-   BAY: RBAM_(—)021020(fni)-   BPU: BPUM_(—)2020(fni)-   OIH: OB0537-   SAU: SA2136(fni)-   SAV: SAV2346(fni)-   SAM: MW2267(fni)-   SAR: SAR2431(fni)-   SAS: SAS2237-   SAC: SACOL2341(fni)-   SAB: SAB2225c(fni)-   SAA: SAUSA300_(—)2292(fni)-   SAO: SAOUHSC_(—)02623-   SEP: SE1925-   SER: SERP1937(fni-2)-   SHA: SH0712(fni)-   SSP: SSP0556-   LMO: lmo1383-   LMF: LMOf2365_(—)1402(fni)-   LIN: lin1420-   LWE: lwe1399(fni)-   LLA: L11083(yebB)-   LLC: LACR_(—)0457-   LLM: llmg_(—)0428(fni)-   SPY: SPy_(—)0879-   SPZ: M5005_Spy_(—)0685-   SPM: spyM18_(—)0940-   SPG: SpyM3_(—)0598-   SPS: SPs1255-   SPH: MGAS10270_Spy0743-   SPI: MGAS10750_Spy0777-   SPJ: MGAS2096_Spy0756-   SPK: MGAS9429_Spy0740-   SPF: SpyM51123(fni)-   SPA: M6_Spy0702-   SPB: M28_Spy0665-   SPN: SP_(—)0384-   SPR: spr0341(fni)-   SPD: SPD_(—)0349(fni)-   SAG: SAG1323-   SAN: gbs1393-   SAK: SAK_(—)1354(fni)-   SMU: SMU.939-   STC: str0562(idi)-   STL: stu0562(idi)-   STE: STER_(—)0601-   SSA: SSA_(—)0336-   SGO: SGO_(—)0242-   LPL: lp_(—)1732(idi1)-   LJO: LJ1208-   LAC: LBA1171-   LSA: LSA0905(idi)-   LSL: LSL_(—)0682-   LDB: Ldb0996(fni)-   LBU: LBUL_(—)0903-   LBR: LVIS_(—)0861-   LCA: LSEI_(—)1493-   LGA: LGAS_(—)1036-   LRE: Lreu_(—)0912-   EFA: EF0901-   OOE: OEOE_(—)1103-   STH: STH1674-   CBE: Cbei_(—)3081-   DRM: Dred_(—)0474-   SWO: Swol_(—)1341-   MTA: Moth_(—)1328-   MTU: Rv1745c(idi)-   MTC: MT1787(idi)-   MBO: Mb1774c(idi)-   MBB: BCG_(—)1784c(idi)-   MPA: MAP3079c-   MAV: MAV_(—)3894(fni)-   MSM: MSMEG_(—)1057(fni) MSMEG_(—)2337(fni)-   MUL: MUL_(—)0380(idi2)-   MVA: Mvan_(—)1582 Mvan_(—)2176-   MGI: Mflv_(—)1842 Mflv_(—)4187-   MMC: Mmcs_(—)1954-   MKM: Mkms_(—)2000-   MJL: Mjls_(—)1934-   CGL: NCg12223(cg12305)-   CGB: cg2531(idi)-   CEF: CE2207-   CDI: DIP1730(idi)-   NFA: nfa19790 nfa22100-   RHA: RHA1_ro00239-   SCO: SCO6750(SC5F2A.33c)-   SMA: SAV1663(idi)-   LXX: Lxx23810(idi)-   CMI: CMM_(—)2889(idiA)-   AAU: AAur_(—)0321(idi)-   PAC: PPA2115-   FRA: Francci3_(—)4188-   FRE: Franean1_(—)5570-   FAL: FRAAL6504(idi)-   KRA: Krad_(—)3991-   SEN: SACE_(—)2627(idiB_(—)2) SACE_(—)5210(idi)-   STP: Strop_(—)4438-   SAQ: Sare_(—)4564 Sare_(—)4928-   RXY: Rxyl_(—)0400-   BBU: BB0684-   BGA: BG0707-   SYN: sll1556-   SYC: syc2161_c-   SYF: Synpcc7942_(—)1933-   CYA: CYA_(—)2395(fni)-   CYB: CYB_(—)2691(fni)-   TEL: t111403-   ANA: a114591-   AVA: Ava_(—)2461 Ava_B0346-   TER: Tery_(—)1589-   SRU: SRU_(—)1900(idi)-   CHU: CHU_(—)0674(idi)-   GFO: GFO_(—)2363(idi)-   FJO: Fjoh_(—)0269-   FPS: FP1792(idi)-   CTE: CT0257-   CCH: Cag_(—)1445-   CPH: Cpha266_(—)0385-   PVI: Cvib_(—)1545-   PLT: Plut_(—)1764-   RRS: RoseRS_(—)2437-   RCA: Rcas_(—)2215-   HAU: Haur_(—)4687-   DRA: DR_(—)1087-   DGE: Dgeo_(—)1381-   TTH: TT_P0067-   TTJ: TTHB110-   MJA: MJ0862-   MMP: MMP0043-   MMQ: MmarC5_(—)1637-   MMX: MmarC6_(—)0906-   MMZ: MmarC7_(—)1040-   MAE: Maeo_(—)1184-   MVN: Mevan_(—)1058-   MAC: MA0604(idi)-   MBA: Mbar_A1419-   MMA: MM_(—)1764-   MBU: Mbur_(—)2397-   MTP: Mthe_(—)0474-   MHU: Mhun_(—)2888-   MLA: Mlab_(—)1665-   MEM: Memar_(—)1814-   MBN: Mboo_(—)2211-   MTH: MTH48-   MST: Msp_(—)0856(fni)-   MSI: Msm_(—)1441-   MKA: MK0776(lldD)-   AFU: AF2287-   HAL: VNG1818G(idi) VNG6081G(crt_(—)1) VNG6445G(crt_(—)2) VNG7060    VNG7149-   HMA: rrnAC3484(idi)-   HWA: HQ2772A(idiA) HQ2847A(idiB)-   NPH: NP0360A(idiB_(—)1) NP4826A(idiA) NP5124A(idiB_(—)2)-   TAC: Ta0102-   TVO: TVN0179-   PTO: PT00496-   PHO: PH1202-   PAB: PAB1662-   PFU: PF0856-   TKO: TK1470-   RCI: LRC397(fni)-   APE: APE_(—)1765.1-   SMR: Smar_(—)0822-   IHO: Igni_(—)0804-   HBU: Hbut_(—)0539-   SSO: SS00063-   STO: ST2059-   SAI: Saci_(—)0091-   MSE: Msed_(—)2136-   PAI: PAE0801-   PIS: Pisl_(—)1093-   PCL: Pcal_(—)0017-   PAS: Pars 0051-   TPE: Tpen_(—)0272    Exemplary Isoprene Synthase Nucleic Acids and Polypeptides    Genbank Accession Nos.-   AY341431-   AY316691-   AY279379-   AJ457070-   AY182241

What is claimed as new and desired to be protected by Letters Patent ofthe United States is:
 1. A method of producing isoprene, the methodcomprising: (a) culturing recombinant cells under suitable cultureconditions for the production of isoprene, wherein the cells comprisenucleic acids encoding (i) a heterologous isoprene synthase polypeptide,(ii) an isopentenyl-diphosphate delta-isomerase (DI) polypeptide, and(iii) at least one of a 1-Deoxyxylulose-5-phosphate synthase(DXS)polypeptide and/or one or more mevalonate (MVA) pathwaypolypeptides and wherein the cells (1) produce greater than about 400nmole/gwcm/hr of isoprene, (2) convert more than about 0.002 molarpercent of the carbon that the cells consume from a cell culture mediuminto isoprene, (3) have an average volumetric productivity of isoprenegreaterthan about 0.1mg/Lbroth/hr of isoprene, or (4) produce at least0.0037 grams of isoprene per gram of dry cell mass (gdcm); and (b)producing isoprene.
 2. The method of claim 1, further comprisingrecovering the isoprene produced by the cells.
 3. The method of claim 1,wherein the cells further comprise an endogenous nucleic acid encodingan isoprene synthase polypeptide.
 4. The method of claim 1, wherein theisoprene synthase polypeptide is a plant isoprene synthase polypeptide.5. The method of claim 4, wherein the plant isoprene synthasepolypeptide is a poplar isoprene synthase polypeptide.
 6. The method ofclaim 4, wherein the plant isoprene synthase polypeptide is a kudzuisoprene synthase polypeptide.
 7. The method of claim 1, wherein thenucleic acid encoding an IDI polypeptide of (a)(ii) is a heterologousnucleic acid encoding an IDI polypeptide.
 8. The method of claim 1,wherein the nucleic acid encoding an IDI polypeptide of (a)(ii) is acopy of an endogenous nucleic acid encoding an IDI polypeptide.
 9. Themethod of claim 1, wherein the nucleic acid encoding a DXS polypeptideof (a)(iii) is a heterologous nucleic acid encoding a DXS polypeptide.10. The method of claim 1, wherein the nucleic acid encoding a DXSpolypeptide of (a)(iii) is a copy of an endogenous nucleic acid encodinga DXS polypeptide.
 11. The method of claim 1, wherein at least one ofthe nucleic acids encoding one or more MVA pathway polypeptides of(a)(iii) is a heterologous nucleic acid.
 12. The method of claim 1,wherein at least one of the nucleic acids encoding one or more MVApathway polypeptides of (a)(iii) is a copy of an endogenous nucleicacid.
 13. The method of claim 1, wherein the cells comprise two or morepolypeptides of the MVA pathway.
 14. The method of claim 1, wherein thecells comprise three or more polypeptides of the MVA pathway.
 15. Themethod of claim 1, wherein the cells comprise four or more polypeptidesof the MVA pathway.
 16. The method of claim 1, wherein the cellscomprise polypeptides of the entire MVA pathway.
 17. The method of claim1, wherein at least one of the nucleic acids encoding a polypeptide of(a)(i)-(a)(iii) is in a vector.
 18. The method of claim 1, wherein atleast one of the nucleic acids encoding a polypeptide of (a)(i)-(a)(iii)is integrated into a chromosome of the cells.
 19. The method of claim 1,wherein at least one of the nucleic acids encoding a polypeptide of(a)(i)-(a)(iii) is over-expressed.
 20. The method of claim 19, whereinthe over-expressed nucleic acid is cloned into a multicopy plasmid. 21.The method of claim 20, wherein the over-expressed nucleic acid isplaced under an inducible promoter or a constitutive promoter.
 22. Themethod of claim 1, wherein the cells are fungal, bacterial, plant, oralgal cells.
 23. The method of claim 22, wherein the cells are fungalcells.
 24. The method of claim 23, wherein the cells are Aspergillus,yeast, or Trichoderma, cells.
 25. The method of claim 24, wherein theyeast cells are Saccharomyces sp., Schizosaccharomyces sp., Pichia sp.,Yarrowia sp. or Candida sp. cells.
 26. The method cells of claim 23,where the cells are selected from the group consisting of A. oryzae, A.niger, S. cerevisiae, S. pombe, T. reesei, H. insolens, H. lanuginose,H. grisea, C. lucknowense, A. sojae, A. japonicus, A. nidulans, A.aculeatus, A. awamori, F. roseum, F. graminum , F. cerealis, F.oxysporuim, F. venenatum, N. crassa, M. miehei, T. viride, F. oxysporum,and F. solan cells.
 27. The method of claim 22, wherein the cells arebacterial cells.
 28. The method of claim 27, wherein the cells aregram-positive bacterial cells or gram-negative bacterial cells.
 29. Themethod of claim 27, wherein the bacterial cells are selected from thegroup consisting of E. coli, P. citrea, B. subtilis, B. licheniformis,B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B.amyloliquefaciens, B. clausii, B. halodurans, B.megaterium, B.coagulans, B. circulans, B. lautus, B. thuringiensis, S. albus, S.lividans, S. coelicolor, S. griseus, Pseudomonas sp., Corynebacteriasp., and P. alcaligenes cells.
 30. The method of claim 22, wherein thecells are plant cells.
 31. The method of claim 30, wherein the plantcells are selected from the group consisting of: the family Fabaceae,the Faboideae subfamily, kudzu, poplar, Populus alba×tremula, Populusalba, aspen, Populus tremuloides, and Quercus robur cells.
 32. Themethod of claim 22, wherein the cells are algal cells.
 33. The method ofclaim 32, wherein the algal cells are selected from the group consistingof: green algae, red algae, glaucophytes, chlorarachniophytes,euglenids, chromista, and dinoflagellates.